Fluid connector

ABSTRACT

Disclosed herein are cell processing systems, devices, and methods thereof. A system for cell processing may comprise a plurality of instruments each independently configured to perform one or more cell processing operations upon a cartridge, and a robot capable of moving the cartridge between each of the plurality of instruments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/987,745, filed Mar. 10, 2020, U.S. Provisional Application No.63/093,038, filed Oct. 16, 2020, the content of each of which is herebyincorporated by reference in its entirety.

TECHNICAL FIELD

Devices, systems, and methods herein relate to manufacturing cellproducts for biomedical applications using automated systems.

BACKGROUND

Cellular therapies based on hematopoietic stem cells (HSCs), chimericantigen receptor (CAR) T cells, NK cells, tumor infiltrating lymphocytes(TILs), T-cell receptors (TCRs), regulatory T cells (T regs), gammadelta (γδ) T cells, and others rely on manufacturing of cell products.Manufacturing of such cell products typically involves multiple cellprocessing steps. Conventional solutions for manufacture of cellproducts rely on cumbersome manual operations performed in expensivebiosafety cabinets and/or clean rooms. Skilled laboratory technicians,adequate sterile enclosures such as cleanroom facilities, and associatedprotocols and procedures for regulated (GMP) manufacturing areexpensive. Many current manufacturing processes employ numerous manualreagent preparation and instrument manipulation steps during amanufacturing protocol, and the processes may require several days oreven weeks. Even platforms described as automated cell processing in aclosed system generally rely on pre-configured instrumentation andtubing sets that limit operational flexibility and do not reliablyprevent process failure due to accidental operator/human error.

Most efforts to automate cell product manufacturing have been directedto automating individual processing steps of a cell therapymanufacturing workflow. Even systems that automate several steps lackend-to-end process flexibility, process robustness, and processscalability. These and other limitations of the previous attempts atautomation of cell processing are addressed in various embodimentsdisclosed here.

SUMMARY

The present disclosure relates generally to methods and systems forprocessing cell products. By processing a cell product in a cartridgemoved between instruments, some variations may achieve one or moreadvantages over prior cell manufacturing systems, including, forexample, improved sterility, automation, lower cost of goods, lowerlabor costs, higher repeatability, higher reliability, lower risk ofoperator error, lower risk of contamination, higher process flexibility,higher capacity, higher instrument throughput, higher degree of processscalability, and shorter process duration. Variations of the disclosuremay comprise a sterile enclosure, thereby reducing the costs ofproviding a clean room environment, and/or utilize a workcell having asmaller footprint than current manufacturing facilities. Furthermore,variations of the methods disclosed herein may, in some cases, beperformed more quickly and with less risk of cell product loss.

In some variations, the disclosure provides a system for cellprocessing, comprising a plurality of instruments each independentlyconfigured to perform one or more cell processing operation upon acartridge, and a robot capable of moving the cartridge between each ofthe plurality of instruments.

In some variations, the system may be enclosed in a workcell. In somevariations, the workcell may be automated. In some variations, theplurality of instruments may be configured to interface with thecartridge to perform cell processing operations upon the cartridge. Insome variations, the system may comprise a processor. The processor maybe configured to control the robot and the plurality of instruments.

In some variations, the system may be configured to receive two or morecartridges. In some variations, the system may comprise the cartridge.In some variations, the cartridge may comprise a plurality of modules.In some variations, the cartridge may comprise a bioreactor module. Insome variations, the cartridge may comprise a cell selection module. Insome variations, the cell selection module may comprise amagnetic-activated cell selection module. In some variations, thecartridge may comprise a sorting module. In some variations, the sortingmodule may comprise a fluorescence activated cell sorting (FACS) module.In some variations, the cartridge may comprise an electroporationmodule. In some variations, the cartridge may comprise a counterflowcentrifugal elutriation (CCE) module.

In some variations, the cartridge may comprise one or more sterileliquid transfer ports. In some variations, the cartridge may comprise aliquid transfer bus fluidically coupled to each module. In somevariations, the cartridge may comprise a pump fluidically coupled to theliquid transfer bus.

In some variations, the system may comprise a pump actuator configuredto interface with the pump. In some variations, the system may comprisea bioreactor instrument. In some variations, the bioreactor instrumentmay comprise multiple slots for cartridges. In some variations, thesystem may comprise a cell selection instrument. In some variations, thecell selection instrument may comprise a magnetic-activated cellselection instrument.

In some variations, the system may comprise a sorting instrument. Insome variations, the sorting instrument may comprise a fluorescenceactivated cell sorting (FACS) instrument. In some variations, the systemmay comprise an electroporation instrument. In some variations, thesystem may comprise a counterflow centrifugal elutriation (CCE)instrument. In some variations, the system may comprise a reagent vault.

In some variations, the cartridge may comprise a bioreactor module and aselection module. In some variations, the cartridge may comprise abioreactor module and a CCE module. In some variations, the cartridgemay comprise a bioreactor module, selection module, and a CCE module. Insome variations, the cartridge may comprise a bioreactor module,selection module, and an electroporation module. In some variations, thecartridge may comprise a bioreactor module, selection module, a CCEmodule, and an electroporation module. In some variations, the cartridgemay comprise a second bioreactor module having an internal volume two ormore, five or more, or ten or more times larger than the internal volumeof the first bioreactor.

In some variations, the system may comprise an enclosure. In somevariations, the enclosure may comprise an ISO7 cleanroom. In somevariations, the enclosure may comprise an ISO6 cleanroom. In somevariations, the enclosure may comprise an ISO5 cleanroom. In somevariations, the enclosure may comprise a feedthrough. In somevariations, the system may perform automated manufacturing of cellproducts.

In some variations, the disclosure provides a cartridge for cellprocessing, comprising a liquid transfer bus and a plurality of modules,each module fluidically coupled to the liquid transfer bus.

In some variations, the cartridge may comprise one or more sterileliquid transfer ports. In some variations, the cartridge may comprise abioreactor module. In some variations, the cartridge may comprise a cellselection module. In some variations, the cell selection module maycomprise a magnetic-activated cell selection module. In some variations,the cartridge may comprise a sorting module. In some variations, thesorting module may comprise a fluorescence activated cell sorting (FACS)module. In some variations, the cartridge may comprise anelectroporation module. In some variations, the cartridge may comprise acounterflow centrifugal elutriation (CCE) module.

In some variations, the cartridge may comprise a mechanoporation module.In some variations, the cartridge may comprise a second bioreactormodule having an internal volume two or more, five or more, or ten ormore times larger than the internal volume of the first bioreactor. Insome variations, the cartridge may comprise a bioreactor module,selection module, and a CCE module. In some variations, the cartridgemay comprise a bioreactor module, selection module, and anelectroporation module. In some variations, the cartridge may comprise abioreactor module, selection module, a CCE module, and anelectroporation module.

In some variations, the disclosure provides a method for processingcells, comprising moving a cartridge containing a cell product between aplurality of instruments inside an enclosed and automated workcell. Theinstruments may interface with the cartridge to perform cell processingsteps on the cell product.

In some variations, cell processing steps may be performed on the cellproduct. In some variations, for each cell product, all cell processingsteps in the method are performed in a single cartridge.

In some variations, the cell product may be split into a plurality ofcell product portions. In some variations, the cell processing steps maybe performed on the plurality of cell product portions in parallel. Insome variations, at least two cell product portions of the plurality ofcell product portions may be combined.

In some variations, the workcell may comprise a robot configured to movecartridges. In some variations, the workcell may comprise a processor.The processor may be configured to control the robot and the pluralityof instruments. In some variations, the workcell may be configured toreceive two or more cartridges.

In some variations, the cartridge may comprise a plurality of modules.In some variations, the cartridge may comprise a bioreactor module. Insome variations, the cartridge may comprise a cell selection module. Insome variations, the cell selection module may comprise amagnetic-activated cell selection module.

In some variations, the cartridge may comprise a sorting module. In somevariations, the sorting module may comprise a fluorescence activatedcell sorting (FACS) module. In some variations, the cartridge maycomprise an electroporation module. In some variations, the cartridgemay comprise a counterflow centrifugal elutriation (CCE) module. In somevariations, the cartridge may comprise one or more sterile liquidtransfer ports. In some variations, the cartridge may comprise a liquidtransfer bus fluidically coupled to each module. In some variations, thecartridge may comprise a pump fluidically coupled to the liquid transferbus.

In some variations, the workcell may comprise a pump actuator configuredto interface with the pump. In some variations, the workcell maycomprise a bioreactor instrument. In some variations, the bioreactorinstrument may comprise multiple slots for cartridges. In somevariations, the method may comprise performing the cell processing stepson two or more cartridges in parallel.

In some variations, the workcell may comprise a cell selectioninstrument. In some variations, the cell selection instrument maycomprise a magnetic-activated cell selection instrument.

In some variations, the workcell may comprise a sorting instrument. Insome variations, the sorting instrument may comprise a fluorescenceactivated cell sorting (FACS) instrument. In some variations, theworkcell may comprise an electroporation instrument. In some variations,the workcell may comprise a counterflow centrifugal elutriation (CCE)instrument. In some variations, the workcell may comprise a reagentvault.

In some variations, the cartridge may comprise a bioreactor module and aselection module. In some variations, the cartridge may comprise abioreactor module and a CCE module. In some variations, the cartridgemay comprise a bioreactor module, selection module, and a CCE module. Insome variations, the cartridge may comprise a bioreactor module,selection module, and an electroporation module. In some variations, thecartridge may comprise a bioreactor module, selection module, a CCEmodule, and an electroporation module.

In some variations, the workcell may comprise an enclosure. In somevariations, the enclosure may comprise an ISO7 cleanroom. In somevariations, the enclosure may comprise an ISO6 cleanroom. In somevariations, the enclosure may comprise an ISO5 cleanroom. In somevariations, the enclosure may comprise a feedthrough.

In some variations, the method may perform automated manufacturing of acell product. In some variations, the cell product may comprise achimeric antigen receptor (CAR) T cell product. In some variations, thecell product may comprise a natural killer (NK) cell product. In somevariations, the cell product may comprise a hematopoietic stem cell(HSC) cell product. In some variations, the cell product may comprise atumor infiltrating lymphocyte (TIL) cell product. In some variations,the cell product may comprise a regulatory T (Treg) cell product.

In some variations, the disclosure provides a method for processing asolution containing a cell product, performed in an automated system,the method comprising one or more cell processing steps, performedserially in any order, selected from: an enrichment step, aconcentration step, a buffer exchange step, a formulation step, awashing step, a selection step, a resting step, an expansion step, atissue-digestion step, an activation step, a transduction step, atransfection step, and a harvesting step.

In some variations, an enrichment step may comprise enriching a selectedpopulation of cells in the solution by conveying the solution to a CCEmodule of the cartridge via a liquid transfer bus, operating the robotto move the cartridge to a CCE instrument so that the CCE moduleinterfaces with the CCE instrument, and operating the CCE instrument tocause the CCE module to enrich the selected population of cells.

In some variations, a washing step may comprise washing a selectedpopulation of cells in the solution by conveying the solution to the CCEmodule of the cartridge via the liquid transfer bus, operating the robotto move the cartridge to the CCE instrument so that the CCE moduleinterfaces with the CCE instrument, and operating the CCE instrument tocause the CCE module to remove media from the solution, introduce mediainto the solution, and/or replace media in the solution.

In some variations, a selection step may comprise selecting a selectedpopulation of cells in the solution by conveying the solution to aselection module of the cartridge via the liquid transfer bus, operatingthe robot to move the cartridge to a selection instrument so that theselection module interfaces with the selection instrument, and operatingthe selection instrument to cause the selection module to select theselected population of cells.

In some variations, a sorting step may comprise sorting a population ofcells in the solution by conveying the solution to a sorting module ofthe cartridge via the liquid transfer bus, operating the robot to movethe cartridge to a sorting instrument so that the sorting moduleinterfaces with the sorting instrument, and operating the sortinginstrument to cause the sorting module to sort the population of cells.

In some variations, a resting step may comprise conveying the solutionto a bioreactor module of the cartridge via the liquid transfer bus,operating the robot to move the cartridge to the bioreactor instrumentso that the bioreactor module interfaces with the bioreactor instrument,and operating the bioreactor instrument to cause the bioreactor moduleto maintain the cells.

In some variations, an expansion step may comprise expanding the cellsin the solution by conveying the solution to the bioreactor module ofthe cartridge via the liquid transfer bus, operating the robot to movethe cartridge to the bioreactor instrument so that the bioreactor moduleinterfaces with the bioreactor instrument, and operating the bioreactorinstrument to cause the bioreactor module to allow the cells to expandby cellular replication.

In some variations, a tissue-digestion step may comprise conveying anenzyme reagent via the liquid transfer bus to a module containing asolution containing a tissue such that the enzyme reagent causesdigestion of the tissue to release a select cell population into thesolution.

In some variations, an activating step may comprise activating aselected population of cells in the solution by conveying an activatingreagent via the liquid transfer bus to a module containing the solutioncontaining the cell product.

In some variations, an electroporation step may comprise conveying thesolution to an electroporation module of the cartridge via the liquidtransfer bus, operating the robot to move the cartridge to anelectroporation instrument so that the electroporation module interfaceswith the electroporation instrument, and operating the electroporationinstrument to cause the electroporation module to electroporate theselected population of cells in the presence of the vector.

In some variations, a transduction step may comprise conveying aneffective amount of a vector via the liquid transfer bus to a modulecontaining the solution containing the cell product, thereby transducinga selected population of cells in the solution. In some variations, afill/finishing step may comprise conveying a formulation solution viathe liquid transfer bus to a module containing the cell product togenerate a finished cell product and conveying the finished cell productto one or more product collection bags.

In some variations, the method may comprise sterilizing, either manuallyor automatically, the cartridge in a feedthrough port. In somevariations, the method may comprise introducing, either manually orautomatically, one or more of a fluid and the cell product into thecartridge via a sterile liquid transfer port. In some variations, themethod may comprise a harvesting step comprising removing, eithermanually or automatically, the cell product from the cartridge. In somevariations, the cell product may comprise an immune cell. In somevariations, in order, the enrichment step, the selection step, theactivation step, the transduction step, the expansion step, and theharvesting step.

In some variations, the immune cell may comprise a geneticallyengineered chimeric antigen receptor T cell. In some variations, theimmune cell may comprise a genetically engineered T cell receptor (TCR)cell. In some variations, the immune cell may comprise is anatural-killer (NK) cell. In some variations, the cell product maycomprise a hematopoietic stem cell (HSC). In some variations, the methodmay comprise, in order, the enrichment step, the selection step, theresting step, the transduction step, and the harvesting step. In somevariations, the cell product may comprise a tumor infiltratinglymphocyte (TIL). In some variations, the method may comprise, in order,the tissue-digestion step, the washing step, the activation step, theexpansion step, and the harvesting step.

Also described here is a counterflow centrifugal elutriation (CCE)module, comprising a conical element having an internal surface and anexternal surface fixedly attached to a distal end of a linear memberhaving an internal surface and an external surface, the proximal end ofthe linear member rotationally attached to a fulcrum to permitextension, retraction, and rotation of the linear member.

Also described here is a workcell comprising an enclosure, a pluralityof instruments each independently configured to perform one or more cellprocessing operation upon a cartridge, and a robot capable of moving thecartridge between each of the plurality of instruments.

In some variations, the enclosure may comprise an air filtration inletconfigured to maintain ISO 7 or better air quality within an interiorzone of the workcell. In some variations, the workcell may be automated.In some variations, the instruments may interface with the cartridge toperform cell processing operations upon the cartridge. In somevariations, the workcell may comprise a processor. The processor may beconfigured to control the robot and the plurality of instruments.

In some variations, the workcell may be configured to receive two ormore cartridges. In some variations, the workcell may comprise thecartridge. In some variations, the cartridge may comprise a plurality ofmodules. In some variations, the cartridge may comprise a bioreactormodule. In some variations, the cartridge may comprise a cell selectionmodule. In some variations, the cell selection module may comprise amagnetic-activated cell selection module. In some variations, thecartridge may comprise a sorting module.

In some variations, the sorting module may comprise a fluorescenceactivated cell sorting (FACS) module. In some variations, the cartridgemay comprise an electroporation module. In some variations, thecartridge may comprise a counterflow centrifugal elutriation (CCE)module. In some variations, the cartridge may comprise one or moresterile liquid transfer ports. In some variations, the cartridge maycomprise a liquid transfer bus fluidically coupled to each module. Insome variations, the cartridge may comprise a pump fluidically coupledto the liquid transfer bus.

In some variations, the workcell may comprise a pump actuator configuredto interface with the pump. In some variations, the workcell maycomprise a bioreactor instrument. In some variations, the bioreactorinstrument may comprise multiple slots for cartridges. In somevariations, the workcell may comprise a cell selection instrument. Insome variations, the cell selection instrument may comprise amagnetic-activated cell selection instrument. In some variations, theworkcell may comprise a sorting instrument. In some variations, thesorting instrument may comprise a fluorescence activated cell sorting(FACS) instrument. In some variations, the workcell may comprise anelectroporation instrument.

In some variations, the workcell may comprise a counterflow centrifugalelutriation (CCE) instrument. In some variations, the workcell maycomprise a reagent vault. In some variations, the cartridge may comprisea bioreactor module and a selection module. In some variations, thecartridge may comprise a bioreactor module and a CCE module. In somevariations, the cartridge may comprise a bioreactor module, selectionmodule, and a CCE module. In some variations, the cartridge may comprisea bioreactor module, selection module, and an electroporation module. Insome variations, the cartridge may comprise a bioreactor module,selection module, a CCE module, and an electroporation module. In somevariations, the cartridge may comprise a second bioreactor module havingan internal volume two or more, five or more, or ten or more timeslarger than the internal volume of the first bioreactor. In somevariations, the enclosure may comprise a feedthrough. In somevariations, the workcell may perform automated manufacturing of cellproducts. In some variations, the system may comprise a plurality ofbioreactor instruments. Each bioreactor instrument may be configured toreceive a single cartridge.

Also described here is a rotor comprising a first side comprising afirst fluid conduit, a second side comprising a second fluid conduit,the second side opposite the first side, and a cone coupled between thefirst fluid conduit and the second fluid conduit.

In some variations, the cone may comprise a bicone. In some variations,the bicone may comprise a first cone including a first base and a secondcone including a second base. The first base may face the second base.In some variations, the rotor may comprise a magnetic portion. In somevariations, the rotor may define a rotation axis. In some variations, atleast a portion of the first fluid conduit and at least a portion of thesecond fluid conduit may extend parallel to the rotation axis. In somevariations, at least a portion of the first fluid conduit and at least aportion of the second fluid conduit may be co-axial.

In some variations, the cone may comprise a volume of between about 10ml and about 40 ml. In some variations, the cone may comprise a coneangle of between about 30 degrees and about 60 degrees. In somevariations, at least a portion of the rotor may be opticallytransparent. In some variations, the rotor may comprise an asymmetricshape. In some variations, a first portion may comprise the cone and asecond portion comprising a paddle shape.

In some variations, a cartridge for cell processing may comprise aliquid transfer bus and a plurality of modules. Each module may befluidically linked to the liquid transfer bus. The cartridge maycomprise a counterflow centrifugal elutriation (CCE) module comprisingthe rotors described herein.

Also described here is a rotor comprising a first fluid conduit, a firstfluid conduit, a first cone coupled to the first fluid conduit. Thefirst cone may comprise a first volume. A second fluid conduit may becoupled to the first cone. A second cone may be coupled to the secondconduit. The second cone may comprise a second volume larger than thefirst volume. A third fluid conduit may be coupled to the second cone.

In some variations, the first cone may comprise a first bicone and thesecond cone may comprise a second bicone. In some variations, the firstbicone may comprise a third cone including a first base and a fourthcone including a second base. The first base may face the second base.The second bicone may comprise a fifth cone including a third base and asixth cone including a fourth base. The third base may face the fourthbase.

In some variations, the rotor may comprise a magnetic portion. In somevariations, at least a portion of the rotor may be opticallytransparent. In some variations, the first fluid conduit may comprise aninlet and the third fluid conduit comprises an outlet.

Also described here is a system for cell processing comprising acartridge comprising a housing comprising a rotor configured to separatecells from a fluid, and an instrument comprising a magnet configured tointerface with the cartridge to magnetically rotate the rotor.

In some variations, the cartridge may be configured to move between aplurality of instruments. In some variations, an air gap may be betweenthe housing and the magnet. In some variations, the housing may enclosethe rotor. In some variations, the housing may comprise a consumablecomponent and the magnet comprises a durable component.

In some variations, the magnet may be releasably coupled to the housing.In some variations, the magnet may be configured to be moved relative tothe housing. In some variations, the separated cells may comprise afirst size and a first density and non-separated cells of the fluidcomprise a second size and a second density different from the firstsize and the first density. Also described here is a cartridge for cellprocessing, comprising a liquid transfer bus and a plurality of modules.Each module may be fluidically linked to the liquid transfer bus. Thecartridge may comprise a counterflow centrifugal elutriation (CCE)module comprising the rotor described here.

Also described here is a method of counterflow centrifugal elutriation(CCE) comprising moving a rotor towards a magnet, the rotor defining arotational axis, flowing the fluid through the rotor, magneticallyrotating the rotor about the rotational axis using the magnet whileflowing the fluid through the rotor.

In some variations, image data of one or more of the fluid and particlesin the rotor may be generated using an optical sensor. One or more of arotation rate of the rotor and a flow rate of the fluid may be selectedbased at least in part on the image data.

In some variations, one or more of the fluid and the cells may beilluminated using an illumination source. In some variations, the methodmay comprise moving the rotor away from the magnet. In some variations,the method may comprising moving the rotor towards an illuminationsource and an optical sensor, and moving the rotor away from theillumination source and the optical sensor.

In some variations, moving the rotor comprises advancing and withdrawingthe magnet relative to the rotor using a robot. In some variations,rotating the rotor comprises a rotation rate of up to 6,000 RPM. In somevariations, flowing the fluid comprises a flow rate of up to about 150ml/min while rotating the rotor.

Also described here is a method of magnetic-activated cell selectioncomprising flowing the fluid comprising input cells into a flow cell. Aset of the cells may be labeled with magnetic-activated cell selection(MACS) reagent. The set of cells may be magnetically attracted towards amagnet array for a dwell time. The set of cells may flow out of the flowcell after the dwell time.

In some variations, the method may comprise incubating the MACS reagentwith the input cells to label the set of cells with the MACS reagent. Insome variations, the method may comprise incubating the MACS reagent maycomprise a temperature between about 1° C. and about 10° C. In somevariations, the method may comprise flowing the set of cells out of theflow cell may comprise flowing a gas through the flow cell. In somevariations, the method may comprise flowing the fluid without the set ofcells out of the flow cell after the dwell time. In some variations, thedwell time may be at least about one minute. In some variations, themagnet array may be disposed external to the flow cell. In somevariations, the method may comprise moving the magnet array relative tothe flow cell. In some variations, moving the magnet array may comprisemoving the magnet array away from the flow cell to facilitate flowingthe set of cells out of the flow cell. In some variations, alongitudinal axis of the flow cell may be perpendicular to ground. Insome variations, the flow cell may be absent beads.

Also described here is a magnetic-activated cell selection (MACS) modulecomprising a flow cell comprising an elongate cavity having a cavityheight, a magnet array may comprise a plurality of magnets. Each of themagnets may be spaced apart by a spacing distance. A ratio of the cavityheight to the spacing distance may be between about 20:1 and about 1:20.

In some variations, the flow cell may comprise a set of linear channelscomprising a first channel parallel to a second channel, and a thirdchannel in fluid communication with each of the first channel and thesecond channel. In some variations, the first channel may comprise afirst cavity height and the second channel may comprise a second cavityheight. A ratio of the first cavity height to a second cavity height maybe between about 1:1 to about 3:7. In some variations, the third channelmay comprise a ratio of a length of the third channel to a diameter ofthe third channel of between about 2:1 to about 6:1.

In some variations, a first fluid conduit may be coupled to an inlet ofthe flow cell and an outlet of the flow cell. The first fluid conduitmay be configured to receive the set of cells from the flow cell. Asecond fluid conduit may be coupled to the inlet of the flow cell andthe outlet of the flow cell. The second fluid conduit may be configuredto receive a fluid without the set of cells from the flow cell.

In some variations, a cartridge for cell processing may comprise aliquid transfer bus and a plurality of modules. Each module may befluidically linked to the liquid transfer bus. The cartridge maycomprise a magnetic-activated cell selection (MACS) module as describedherein.

Also described here is a system for cell processing comprising acartridge comprising a rotor configured for counterflow centrifugalelutriation of cells in a fluid. A first magnet may be configured tomagnetically rotate the rotor and separate the cells from the fluid inthe rotor. The cartridge may further comprise a flow cell in fluidcommunication with the rotor and configured to receive the cells fromthe rotor. A second magnet may be configured to magnetically separatethe cells in the flow cell.

In some variations, an illumination source may be configured toilluminate the cells. An optical sensor may be configured to generateimage data corresponding to the cells. In some variations, the systemmay comprise one or more of an oxygen depletion sensor, leak sensor,inertial sensor, pressure sensor, and bubble sensor. In some variations,the system may comprise one or more valves and pumps.

In some variations, the separated cells may comprise a first size and afirst density and non-separated cells of the fluid comprise a secondsize and a second density different from the first size and the firstdensity.

Also described here is an electroporation module comprising a fluidconduit configured to receive a first fluid comprising cells and asecond fluid, a set of electrodes coupled to the fluid conduit, a pumpcoupled to the fluid conduit, andd a controller comprising a processorand memory. The controller may be configured to generate a first signalto introduce the first fluid into the fluid conduit using the pump,generate a second signal to introduce the second fluid into the fluidconduit such that the second fluid separates the first fluid from athird fluid, and generate an electroporation signal to electroporate thecells in the fluid conduit using the set of electrodes.

In some variations, the second fluid may comprise a gas or oil. In somevariations, the controller may be configured to generate a third signalto introduce the third fluid into the fluid conduit, the third fluidseparated from the first fluid by the second fluid.

In some variations, a cartridge for cell processing may comprise aliquid transfer bus and a plurality of modules. Each module may befluidically linked to the liquid transfer bus. The cartridge maycomprise an electroporation module as described here.

Also described here is a method of electroporating cells comprisingreceiving a first fluid comprising cells in a fluid conduit, receiving asecond fluid in the fluid conduit to separate the first fluid from athird fluid, and applying an electroporation signal to the first fluidto electroporate the cells.

In some variations, the method may comprise receiving the third fluid inthe fluid conduit separated from the first fluid by the second fluid. Insome variations, the first fluid substantially static when applying theelectroporation signal.

Also described here is a method of electroporating cells comprisingreceiving a first fluid comprising cells in a fluid conduit, applying aresistance measurement signal to the first fluid using a set ofelectrodes, measuring a resistance between the first fluid and the setof electrodes, and applying an electroporation signal to the first fluidbased on the measured resistance.

In some variations, the method may comprise receiving a second fluidcomprising a gas in the fluid conduit before applying theelectroporation signal to the fluid, the first fluid separated from athird fluid by the second fluid.

Also described here is a bioreactor comprising an enclosure comprising abase, a top, and at least one sidewall. A gas-permeable membrane may becoupled to one or more of the base and the sidewall of the enclosure.

In some variations, the enclosure may comprise one or more nestedsurfaces curved around a longitudinal axis of the enclosure. In somevariations, the one more nested surfaces may comprise a set ofconcentric toroids. In some variations, the enclosure may comprise atoroid shape. In some variations, the enclosure may comprise a firstchamber having a first volume and a second chamber having a secondvolume, the first chamber separated from the second chamber, and thefirst volume smaller than the second volume. In some variations, theenclosure may comprise a column extending along a longitudinal axis ofthe enclosure. In some variations, a cavity may be between the enclosureand the gas-permeable membrane. In some variations, the gas-permeablemembrane may extend along the base and the sidewall of the enclosure. Insome variations, an outer surface of the gas-permeable membrane maycomprise one or more projections.

In some variations, a base of the gas-permeable membrane may comprise anangle between about 3 degrees and about 10 degrees relative to the baseof the enclosure. In some variations, the gas-permeable membrane maycomprise a curved surface. In some variations, the gas-permeablemembrane may comprise a set of patterned curved surfaces. In somevariations, the set of patterned curved surfaces may comprise a radiusof curvature of between about 50 mm and about 500 mm.

In some variations, a cartridge for cell processing may comprise aliquid transfer bus and a plurality of modules. Each module may befluidically linked to the liquid transfer bus. The cartridge maycomprise a bioreactor module as described here. In some variations, asystem for cell processing may comprising the cartridge described hereand may further comprise a bioreactor instrument configured to interfacewith the cartridge. The bioreactor instrument may comprise an agitatorconfigured to couple to the bioreactor. The agitator may be configuredto agitate cell culture media comprising cells. In some variations, afluid connector may be configured to couple the bioreactor to a liquidtransfer bus. The fluid connector may comprise foldable sidewalls. Insome variations, the system may comprise a temperature regulator coupledto the bioreactor. In some variations, the system may comprise a gasregulator coupled to the bioreactor.

Also described here is a fluid connector comprising a first connectorcomprising a first proximal end configured to couple to a first fluiddevice, and a first distal end comprising a first port. A secondconnector may comprise a second proximal end configured to couple to asecond fluid device, and a second distal end comprising a second portconfigured to couple to the first port. The first distal end maycomprise a first lumen and the second distal end may comprise a secondlumen. One of the first valve and the second valve may be configured totranslate within the first lumen and the second lumen.

In some variations, the first valve and the second valve may beconfigured to transition from a closed configuration to an openconfiguration only when the first valve couples to the second valve. Insome variations, the first port and the second port may be configured totransition between an open configuration and a closed configuration. Insome variations, the first connector may comprise a first port actuatorand/or the second connector comprises a second port actuator. In somevariations, the second port may be coupled to the first port defines achamber.

In some variations, one or more of the first connector and the secondconnector may comprise a sterilant port configured to couple to asterilant source. The sterilant port may be configured to be in fluidcommunication with the first distal end and the second distal end whenthe second port is coupled to the first port.

In some variations, the chamber may be configured to receive one or moreof a fluid and a sterilant from the sterilant port. In some variations,the sterilant port may be configured to receive a sterilant such thatthe sterilant sterilizes the first connector and the second connector.

In some variations, the first connector may comprise a first valve, andthe second connector may comprise a second valve configured to couple tothe first valve. In some variations, a first seal may comprise the firstport coupled to the second port, and a second seal may comprise thefirst valve coupled to the second valve. In some variations, thesterilant may comprise one or more of vaporized hydrogen peroxide andethylene oxide.

In some variations, the fluid connector may comprise one or more robotengagement features. In some variations, the first connector maycomprise a first alignment feature and the second connector may comprisea second alignment feature configured to couple to the first alignmentfeature in a predetermined axial and rotational configuration. In somevariations, one or more of the first fluid device and the second fluiddevice may comprise an instrument.

In some variations, a system may further comprise a robot configured tooperate the fluid connector, and a controller comprising a memory andprocessor. The controller may be coupled to the robot. The controllermay be configured to generate a first port signal to couple the firstport to the second port using the robotic arm. In some variations, thecontroller may be configured to generate a first valve signal totranslate the first valve relative to the second valve using the roboticarm, and generate a second valve signal to transition the first valveand the second valve to the open configuration. In some variations, thecontroller may be configured to generate a second port signal todecouple the first port from the second port. A sterility of the fluidconnector may be maintained before coupling the first port to the secondport and after decoupling the first port from the second port.

In some variations, a fluid pump may be coupled to the sterilant source.The controller may be configured to generate a first fluid pump signalto circulate a fluid into the chamber through the sterilant port. Insome variations, the controller may be configured to generate a secondfluid pump signal to circulate the sterilant into the chamber throughthe sterilant port to sterilize at least the chamber.

In some variations, the controller may be configured to generate a thirdfluid pump signal to remove the sterilant from the chamber. In somevariations, the controller may be configured to generate a thermalsterilization signal to thermally sterilize the fluid connector. In somevariations, the controller may be configured to generate a radiationsterilization signal to sterilize the fluid connector using radiation.In some variations, the robot may be configured to couple a fluidconnector between at least two of the plurality of instruments and thecartridge.

In some variations, the fluid connector may further comprise acontroller comprising a memory and processor, the controller coupled tothe robot. The controller may be configured to generate a port signal tocouple the first port to the second port using the robotic arm, generatea first valve signal to translate the first valve relative to the secondvalve using the robotic arm, and generate a second valve signal totransition the first valve and the second valve to the openconfiguration.

Also described here is a non-transitory computer-readable medium fortransforming user-defined cell processing operations into cellprocessing steps to be executed by an automated cell processing system.The non-transitory computer-readable medium may comprise instructionsstored thereon that when executed on a processor perform the steps ofreceiving an ordered input list of cell processing operations, andexecuting a transformation model on the ordered input list to create anordered output list of cell processing steps capable of being performedby the system.

In some variations, the ordered output list may be capable of beingperformed by the system to control a robot to move one or morecartridges each containing a cell product between the instruments, andcontrol the instruments to perform cell processing steps on each cellproduct.

In some variations, the method may comprise receiving one or more setsof cell processing parameters, each set associated with one of the cellprocessing operations, and each set of cell processing parametersspecifying characteristics of the cell processing step to be performedby the instrument at that cell processing step. In some variations, thetransformation model may comprise constraints on the ordered output listdetermined by configuration of the automated cell processing system. Insome variations, the constraints may comprise information on theconfiguration of the automated cell processing system. In somevariations, the constraints may comprise one or more of a type and/or anumber of instruments, a type and/or a number of modules on thecartridge, a type and a number of reservoirs on the cartridge, a typeand/or a number of sterile liquid transfer ports on the cartridge, and anumber and a position of fluid paths between the modules, reservoirs,and sterile liquid transfer ports on the cartridge.

In some variations, the steps may further comprise receiving a set ofmore than one ordered input lists of cell processing operations to beperformed on more than one cartridge on the automated cell processingsystem, and executing the transformation model on the sets of orderedinput lists to create the ordered output list of cell processing steps.The ordered output list may be capable of being executed by the systemto control the robot to move the more than one cartridges, eachcomprising its cell product, between the instruments, and control theinstruments to perform cell processing steps on each cell product ofeach cartridge.

In some variations, an automated cell processing system may comprise thenon-transitory computer-readable medium of any preceding claim.

In some variations, a computer-implemented method for transforminguser-defined cell processing operations into cell processing steps to beexecuted by a processor of an automated cell processing system maycomprise receiving an ordered input list of cell processing operations,and executing a transformation model on the ordered input list to createan ordered output list of cell processing steps capable of beingperformed by the system.

In some variations, the method may include controlling a robot to moveone or more cartridges each containing a cell product between theinstruments, and controlling the instruments to perform cell processingsteps on each cell product.

In some variations, the method may comprise receiving one or more setsof cell processing parameters, each set associated with one of the cellprocessing operations, and each set of cell processing parametersspecifying characteristics of the cell processing step to be performedby the instrument at that cell processing step. In some variations, thetransformation model may comprise constraints on the ordered output listdetermined by configuration of the automated cell processing system. Insome variations, the constraints may comprise information on theconfiguration of the automated cell processing system.

In some variations, the constraints may comprise one or more of a typeand/or number of instruments, a type and/or number of modules on thecartridge, a type and number of reservoirs on the cartridge, a typeand/or number of sterile liquid transfer ports on the cartridge, and anumber and position of fluid paths between the modules, reservoirs, andsterile liquid transfer ports on the cartridge.

In some variations, the method may comprise receiving a set of more thanone ordered input lists of cell processing operations to be performed onmore than one cartridge on the automated cell processing system,executing the transformation model on the sets of ordered input lists tocreate the ordered output list of cell processing steps, controlling therobot to move the more than one cartridges, each comprising its cellproduct, between the instruments, and controlling the instruments toperform cell processing steps on each cell product of each cartridge.

Additional variations, features, and advantages of the invention will beapparent from the following detailed description and through practice ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a block diagram of an illustrative variation of a cellprocessing system.

FIG. 1B is a block diagram of an illustrative variation of a cartridge.

FIG. 2A is a block diagram of an illustrative variation of a cellprocessing system. FIG. 2B is a perspective view of an illustrativevariation of a workcell of a cell processing system. FIG. 2C is aperspective view of an illustrative variation of a workcell andcartridge of a cell processing system. FIG. 2D is a block diagram of anillustrative variation of a cell processing system. FIG. 2E is a blockdiagram of another illustrative variation of a cell processing system.

FIG. 3 is a block diagram of another illustrative variation of a cellprocessing system.

FIG. 4A is a perspective view of another illustrative variation of acell processing system.

FIG. 4B is another perspective view of another illustrative variation ofa cell processing system.

FIG. 5 is a perspective view of another illustrative variation of a cellprocessing system.

FIG. 6 is a schematic diagram of an illustrative variation of acartridge.

FIG. 7 is a schematic diagram of another illustrative variation of acartridge.

FIG. 8A is a side view of an illustrative variation of a cartridge. FIG.8B is a top view of an illustrative variation of a cartridge. FIG. 8C isa side view of an illustrative variation of a cartridge. FIG. 8D is aperspective view of an illustrative variation of a cartridge.

FIG. 9 shows a cross-sectional side view of an illustrative variation ofa cartridge.

FIG. 10A shows an illustrative variation of a rotary valve and anactuator. FIG. 10B shows an illustrative variation of a rotary valvedocked with an actuator.

FIG. 11A is a perspective view of an illustrative variation of acartridge comprising a CCE module in an extended configuration. FIG. 11Bis a cross-sectional side view of illustrative variation of a CCE modulein a retracted configuration. FIG. 11C is a cross-sectional side view ofan illustrative variation of a CCE module in an extended configuration.

FIG. 12A is a perspective view of an illustrative variation of amagnetic-activated cell sorting (MACS) instrument comprising a magnet inan ON configuration. FIG. 12B is a perspective view of an illustrativevariation of a MACS instrument comprising a magnet in an OFFconfiguration.

FIG. 13A is a perspective view of an illustrative variation of acartridge and a bioreactor instrument. FIG. 13B is a perspective view ofan illustrative variation of a cartridge coupled to a bioreactorinstrument.

FIG. 14 is a perspective view of an illustrative variation of abioreactor instrument comprising a set of cartridges and cavitiesconfigured to receive cartridges.

FIG. 15 is a block diagram of an illustrative variation of a fluidconnector system.

FIG. 16A is a schematic diagram of an illustrative variation of a fluidconnector. FIG. 16B is a detailed schematic diagram of the fluidconnector depicted in FIG. 16A. FIG. 16C is a schematic diagram of thefluid connector depicted in FIG. 16A in a coupled configuration. FIG.16D is a schematic diagram of the fluid connector depicted in FIG. 16Ain an open port configuration. FIG. 16E is a schematic diagram of thefluid connector depicted in FIG. 16A receiving a gas. FIG. 16F is aschematic diagram of the fluid connector depicted in FIG. 16A receivinga sterilant. FIG. 16G is a schematic diagram of the fluid connectordepicted in FIG. 16A in an open valve configuration. FIG. 16H is aschematic diagram of the fluid connector depicted in FIG. 16Atransferring fluid between fluid devices coupled to the fluid connector.FIG. 16I is a schematic diagram of the fluid connector depicted in FIG.16A in a closed valve configuration. FIG. 16J is a schematic diagram ofthe fluid connector depicted in FIG. 16A in a closed port configuration.FIG. 16K is a schematic diagram of the fluid connector depicted in FIG.16A in an uncoupled configuration. FIG. 16L is a schematic diagram ofthe fluid connector depicted in FIG. 16A uncoupled from a sterilantsource.

FIG. 17A is a front perspective view of a fluid connector in a closedport configuration. FIG. 17B is a rear perspective view of the fluidconnector depicted in FIG. 17A in the closed port configuration. FIG.17C is a rear view of the fluid connector depicted in FIG. 17B in theclosed port configuration. FIG. 17D is a front perspective view of afluid connector in an open port configuration. FIG. 17E is a rearperspective view of the fluid connector depicted in FIG. 17D in the openport configuration. FIG. 17F is a rear view of the fluid connectordepicted in FIG. 17E in the open port configuration.

FIG. 18A is a side view of a fluid connector in an uncoupledconfiguration. FIG. 18B is a cross-sectional side view of a fluidconnector in an uncoupled configuration. FIG. 18C is a side view of afluid connector in a coupled configuration. FIG. 18D is across-sectional side view of a fluid connector in a coupledconfiguration. FIG. 18E is a side view of a fluid connector in an openport configuration. FIG. 18F is a cross-sectional side view of a fluidconnector in an open port configuration. FIG. 18G is a side view of afluid connector in an open valve configuration. FIG. 18H is across-sectional side view of a fluid connector in an open valveconfiguration.

FIG. 19 is a schematic diagram of an illustrative variation of a fluidconnector system.

FIG. 20A is a schematic diagram of an illustrative variation of a fluidconnector system.

FIGS. 20B and 20C are schematic diagrams of an illustrative variation ofa fluid connector connection process.

FIG. 21 is a block diagram of an illustrative variation of a fluidconnector system.

FIG. 22 is a block diagram of an illustrative variation of a fluidconnector system.

FIG. 23 is a block diagram of an illustrative variation of a fluidconnector system.

FIG. 24A is a block diagram of an illustrative variation of a fluidconnector system. FIG. 24B is a schematic diagram of an illustrativevariation of a fluid connector connection process. FIG. 24C is aschematic diagram of an illustrative variation of a valve.

FIG. 25A is a block diagram of an illustrative variation of a fluidconnector system. FIG. 25B is a schematic diagram of an illustrativevariation of a fluid connector connection process. FIG. 25C is aschematic diagram of an illustrative variation of a valve.

FIG. 26A is a side view of an illustrative variation of a pump actuatorand pump. FIG. 26B is a side view of an illustrative variation of a pumpactuator coupled to a pump.

FIG. 27 is a flowchart of an illustrative variation of a method oftransferring fluid using a fluid connector.

FIG. 28 is a flowchart of an illustrative variation of a method of cellprocessing.

FIG. 29 is a flowchart of an illustrative variation of a method of cellprocessing.

FIG. 30A is a flowchart of an illustrative variation of a method of cellprocessing for autologous CART cells or engineered TCR cells. FIG. 30Bis a flowchart of an illustrative variation of a method of cellprocessing for allogeneic CART cells or engineered TCR cells.

FIG. 31 is a flowchart of an illustrative variation of a method of cellprocessing for HSC cells.

FIG. 32 is a flowchart of an illustrative variation of a method of cellprocessing for TIL cells.

FIG. 33 is a flowchart of an illustrative variation of a method of cellprocessing for NK-CAR cells.

FIGS. 34A-34C are flowcharts of illustrative variations of methods ofcell processing for Tre_(g) cells.

FIG. 35 is a flowchart of an illustrative variation of a method of cellprocessing.

FIG. 36 is a flowchart of an illustrative variation of a method ofexecuting a transformation model.

FIG. 37 is an illustrative variation of a graphical user interfacerelating to an initial process design interface.

FIG. 38 is an illustrative variation of a graphical user interfacerelating to creating a process.

FIG. 39 is an illustrative variation of a graphical user interfacerelating to an empty process.

FIG. 40 is an illustrative variation of a graphical user interfacerelating to adding a reagent and a consumable container.

FIG. 41 is an illustrative variation of a graphical user interfacerelating to a process parameter.

FIG. 42 is an illustrative variation of a graphical user interfacerelating to a patient weight process parameter.

FIG. 43 is an illustrative variation of a graphical user interfacerelating to a preprocess analytic.

FIG. 44 is an illustrative variation of a graphical user interfacerelating to a white blood cell count preprocess analytic.

FIG. 45 is an illustrative variation of a graphical user interfacerelating to process parameter calculation.

FIG. 46 is an illustrative variation of a graphical user interfacerelating to a completed process setup.

FIG. 47 is an illustrative variation of a graphical user interfacerelating to process operations activation settings.

FIG. 48 is an illustrative variation of a graphical user interfacerelating to a filled process operations activation settings.

FIG. 49 is an illustrative variation of a graphical user interfacerelating to an initial process operations.

FIG. 50 is an illustrative variation of a graphical user interfacerelating to dragging in process operations.

FIG. 51 is another illustrative variation of a graphical user interfacerelating to dragging in process operations.

FIG. 52 is an illustrative variation of a graphical user interfacerelating to a filled process operations.

FIG. 53 is an illustrative variation of a graphical user interfacerelating to product monitoring.

FIG. 54 is another illustrative variation of a graphical user interfacerelating to product monitoring.

FIG. 55 is a block diagram of an illustrative variation of amanufacturing workflow.

FIG. 56 is a block diagram of an illustrative variation of a cellseparation system.

FIG. 57 is a cross-sectional side view of an illustrative variation of acounterflow centrifugal elutriation (CCE) module.

FIG. 58 is a cross-sectional side view of an illustrative variation of amagnetic-activated cell selection (MACS) module.

FIGS. 59A-59C are perspective views of an illustrative variation of aCCE system. FIG. 59D is a side cross-sectional view of an illustrativevariation of a CCE system. FIGS. 59E-59G are side cross-sectional viewsof an illustrative variation of a rotor of a CCE module.

FIG. 60A is a plan view of an illustrative variation of a rotor of a CCEmodule. FIGS. 60B and 60C are perspective views of an illustrativevariation of a rotor of a CCE module. FIG. 60D is a side view of anillustrative variation of a rotor of a CCE module. FIG. 60E is aperspective view of an illustrative variation of a rotor in a housing.FIGS. 60F and 60G are plan schematic views of illustrative variations ofa rotor of a CCE module. FIG. 60H is a side view of an illustrativevariation of a rotor of a CCE module. FIG. 60I is a perspective view ofanother illustrative variation of a rotor of a CCE module. FIG. 60J is aperspective view of yet another illustrative variation of a rotor of aCCE module. FIG. 60K is a schematic plan view of another illustrativevariation of rotor dimensions of a CCE module. FIG. 60L is an image of aset of illustrative variations of rotors of a CCE module.

FIGS. 61A-61C are schematic views of an illustrative variation of a cellseparation process.

FIG. 62A is a perspective view of an illustrative variation of a MACSsystem in a first configuration. FIG. 62B is a perspective view of anillustrative variation of a MACS system in a second configuration. FIG.62C is a cross-sectional side view of an illustrative variation of aMACS system. FIG. 62D is a perspective view of an illustrative variationof a MACS system in the second configuration. FIG. 62E is a plan view ofan illustrative variation of a flow cell and magnet array of a MACSsystem. FIG. 62F is a plan view of an illustrative variation of a flowcell of a MACS system. FIG. 62G is a schematic diagram of anillustrative variation of a flow cell and magnet array.

FIGS. 63A-63E are perspective views of illustrative variations of amagnet array.

FIG. 64A is a perspective view of an illustrative variation of a flowcell. FIG. 64B is a cross-sectional side view of an illustrativevariation of a flow cell. FIG. 64C is a schematic diagram of anillustrative variation of a MACS system.

FIGS. 65A-65C are schematic diagrams of an illustrative variation of aflow cell.

FIGS. 66A-66C are schematic diagrams of an illustrative variation of acell separation process.

FIGS. 67A-67D are schematic diagrams of an illustrative variation of acell processing system.

FIG. 68A is a cross-sectional perspective view of an illustrativevariation of a bioreactor. FIG. 68B is a cross-sectional side view of anillustrative variation of a bioreactor. FIG. 68C is a perspective viewof an illustrative variation of an enclosure of a bioreactor. FIG. 68Dis a plan view of an illustrative variation of an enclosure of abioreactor.

FIG. 68E is a perspective view of an illustrative variation of amembrane of a bioreactor. FIG. 68F is a side view of an illustrativevariation of a membrane of a bioreactor. FIG. 68G is a perspective viewof an illustrative variation of a membrane of a bioreactor. FIG. 68H isa bottom view of an illustrative variation of a membrane of abioreactor.

FIG. 69A is a cross-sectional side view of an illustrative variation ofan enclosure of a bioreactor. FIG. 69B is a cross-sectional perspectiveview of an illustrative variation of an enclosure of a bioreactor.

FIG. 70 is an exploded perspective view of an illustrative variation ofa bioreactor.

FIG. 71A is a plan view of an illustrative variation of a bioreactor.FIG. 71B is a cross-sectional side view of an illustrative variation ofa bioreactor.

FIG. 72 is a schematic diagram of an illustrative variation of anelectroporation system.

FIG. 73 is an exploded perspective view of an illustrative variation ofan electroporation module.

FIGS. 74A-74B are schematic diagrams of illustrative variation of anelectroporation process.

FIG. 75 is a circuit diagram of an illustrative variation of anelectroporation process.

FIGS. 76A-76D are plots of illustrative variations of an electroporationprocess.

FIG. 77A is a flowchart of an illustrative variation of a method ofseparating cells. FIG. 77B is a flowchart of an illustrative variationof a method of concentrating cells. FIG. 77C is a flowchart of anillustrative variation of a method of buffer exchange.

FIG. 78 is a flowchart of another illustrative variation of a method ofseparating cells.

FIG. 79A is a flowchart of an illustrative variation of a closed-loopmethod of separating cells 7900. FIG. 79B is a flowchart of anillustrative variation of a closed-loop method of elutriating cells7910. FIG. 79C is a flowchart of an illustrative variation of aclosed-loop method of harvesting cells 7920.

FIG. 80A is a flowchart of an illustrative variation of a method ofseparating cells. FIG. 80B is a flowchart of an illustrative variationof a method of selecting cells.

FIG. 81 is a flowchart of another illustrative variation of a method ofseparating cells.

FIG. 82A is a flowchart of an illustrative variation of a method ofpreparing a bioreactor. FIG. 82B is a flowchart of an illustrativevariation of a method of loading a bioreactor. FIG. 82C is a flowchartof an illustrative variation of a method of preparing a bioreactor. FIG.82D is a flowchart of an illustrative variation of a method ofcalibration for a bioreactor. FIG. 82E is a flowchart of an illustrativevariation of a method of mixing reagents. FIG. 82F is a flowchart of anillustrative variation of a method of mixing reagents. FIG. 82G is aflowchart of an illustrative variation of a method of culturing cells.FIG. 82H is a flowchart of an illustrative variation of a method ofrefrigerating cells. FIG. 82I is a flowchart of an illustrativevariation of a method of taking a sample. FIG. 82J is a flowchart of anillustrative variation of a method of culturing cells. FIG. 82K is aflowchart of an illustrative variation of a method of media exchange.FIG. 82L is a flowchart of an illustrative variation of a method ofcontrolling gas. FIG. 82M is a flowchart of an illustrative variation ofa method of controlling pH.

FIG. 83 is a flowchart of an illustrative variation of a method ofelectroporating cells.

FIG. 84 is a flowchart of another illustrative variation of a method ofelectroporating cells.

FIG. 85 are schematic diagrams of an illustrative variation of a fluidconnector.

FIG. 86 are schematic diagrams of an illustrative variation of a fluidconnector port.

FIG. 87 is a schematic diagram of an illustrative variation of a fluidconnector connection process.

FIG. 88 is a schematic diagram of an illustrative variation of a fluidconnector connection process.

FIG. 89 is a schematic diagram of an illustrative variation of a fluidconnector.

FIG. 90A is a side view of an illustrative variation of a fluidconnector. FIG. 90B is a perspective view of the fluid connectordepicted in FIG. 90A. FIG. 90C is a cross-sectional side view of thefluid connector depicted in FIG. 90A.

FIG. 91A is a side view of an illustrative variation of a fluidconnector. FIG. 91B is a perspective view of the fluid connectordepicted in FIG. 91A. FIG. 91C is a cross-sectional side view of thefluid connector depicted in FIG. 91A.

FIG. 91D is a side view of an illustrative variation of a fluidconnector. FIG. 91E is a perspective view of the fluid connectordepicted in FIG. 91D. FIG. 91F is a cross-sectional side view of thefluid connector depicted in FIG. 91D.

FIG. 92A is a side view of an illustrative variation of a fluidconnector. FIG. 92B is a transparent side view of the fluid connectordepicted in FIG. 92A. FIG. 92C is a perspective view of the fluidconnector depicted in FIG. 92A. FIG. 92D is a cross-sectional side viewof the fluid connector depicted in FIG. 92A.

FIG. 93A is a perspective view of an illustrative variation of a fluidconnector. FIG. 93B is a transparent perspective view of the fluidconnector depicted in FIG. 93A.

FIG. 94A is a perspective view of an illustrative variation of a fluidconnector. FIG. 94B is a transparent perspective view of the fluidconnector depicted in FIG. 94A.

FIG. 95A is a perspective view of an illustrative variation of a fluidconnector. FIG. 95B is a transparent perspective view of the fluidconnector depicted in FIG. 95A. FIG. 95C is a detailed side view of aport in an open port configuration. FIG. 95D is a detailed side view ofa port in a closed port configuration.

FIG. 96A is a plan view of an illustrative variation of a fluid device.FIG. 96B is a side view of an illustrative variation of a fluid devicecoupled to a robot. FIG. 96C is a perspective view of an illustrativevariation of a fluid device held by a robot.

FIG. 97A is a perspective view of an illustrative variation of a MACSmodule. FIG. 97B is a cross-sectional perspective view of anillustrative variation of a MACS module. FIG. 97C is a cross-sectionalside view of an illustrative variation of a MACS module.

FIG. 98 is a flowchart of an illustrative variation of a method of cellprocessing.

FIG. 99 is a flowchart of an illustrative variation of a method of cellprocessing.

FIG. 100 is a flowchart of an illustrative variation of a method of cellprocessing.

FIG. 101 is a flowchart of an illustrative variation of a method of cellprocessing.

FIG. 102 is a schematic diagram of an illustrative variation of a cellprocessing system.

FIGS. 103A and 103B are perspective views of an illustrative variationof a sterile liquid transfer device.

DETAILED DESCRIPTION

Systems and methods for processing and manufacturing cell products forbiomedical applications are described herein. Cell processing methodsand systems may comprise moving a cartridge containing a cell productbetween a plurality of instruments inside a workcell. One or moreinstruments may be configured to interface with the cartridge to performcell processing steps on the cell product, such that the system (e.g.,workcell) performs cell processing steps on the cell product. In somevariations, a plurality of cell processing steps may be performed withina single cartridge. For example, a robotic arm may be configured to movea cartridge between instruments for different cell processing steps. Thecartridge may comprise a plurality of cell processing devices (e.g.,modules) such as a bioreactor, a counterflow centrifugal elutriation(CCE) module, a magnetic cell sorter (e.g., magnetic-activated cellselection module), an electroporation device (e.g., electroporationmodule), a sorting module (e.g. fluorescence activated cell sorting(FACS) module), an acoustic flowcell module, a centrifugation module, amicrofluidic enrichment module, combinations thereof, and the like. Insome variations, the system may process two or more cartridges inparallel. For example, the bioreactor may comprise a plurality of slotsconfigured to interface with a plurality of cartridges concurrently, asone process step (e.g., cell culturing in a bioreactor) may typically bethe rate limiting step for the operation of the cell processing system.The cell processing systems described herein may reduce operatorintervention and increase throughput by automating cartridge (and cellproduct) movement between instruments using a robot. However, in somevariations, the cartridge may be moved between instruments manually.Furthermore, throughput of the system may be increased by using aplurality of bioreactors, thereby allowing the system to simultaneouslyprocess a plurality of cartridges for a plurality of patients. Moreover,the automated cell processing system may facilitate sterile liquidtransfers between the cartridge and instruments or other components ofthe system such as a fluid connector (e.g., sterile liquid transferport), reagent vault, a second cartridge, a sampling vessel (e.g.,sterile liquid transfer device, combinations thereof, and the like.

Workcell

In some variations, a system for cell processing (e.g., workcell) maycomprise a plurality of instruments each independently configured toperform one or more cell processing operations upon a cartridge. A robotmay be configured to move the cartridge between each of the plurality ofinstruments. The instruments may comprise one or more of a bioreactorinstrument, a cell selection instrument (e.g., a magnetic-activated cellselection instrument), a sorting instrument (e.g., a fluorescenceactivated cell sorting (FACS) instrument), an electroporationinstrument, a counterflow centrifugal elutriation (CCE) instrument, areagent vault, and the like. The system may perform automatedmanufacturing of cell products.

A cartridge may be configured to be portable and facilitate automatedand sterile cell processing using a workcell and robot. For example, thecartridge may be configured to move relative to one or more instrumentsof the workcell to perform different cell processing steps. In somevariations, an instrument may be configured to move relative to acartridge. In some variations, the cartridge may comprise a plurality ofmodules including one or more of a bioreactor module, a cell selectionmodule (e.g., magnetic-activated cell selection module), a sortingmodule (e.g., fluorescence activated cell sorting (FACS) module), anelectroporation module, and a counterflow centrifugal elutriation (CCE)module. The cartridge may further comprise one or more of a sterileliquid transfer port, a liquid transfer bus fluidically coupled to eachmodule, and a pump fluidically coupled to the liquid transfer bus.

In some variations, a method of processing a solution containing a cellproduct may include the cell processing steps of digesting tissue usingan enzyme reagent to release a select cell population into solution,enriching cells using a CCE instrument, washing cells using the CCEinstrument, selecting cells in the solution using a selectioninstrument, sorting cells in the solution using a sorting instrument,differentiating or expanding the cells in a bioreactor, activating cellsusing an activating reagent, electroporating cells, transducing cellsusing a vector, and finishing a cell product.

Cell Selection System

The cell processing systems described herein may comprise a cellselection system configured to separate cells based on predeterminedcriteria. For example, cells may be separated based on physicalcharacteristics such as size and/or density using, for example, acounterflow centrifugation elutriation instrument. Cells may also beseparated based on the presence of predetermined antigens of a cellusing, for example, a magnetic-activated cell selection instrument. Insome variations, a cell selection system comprising modules for theseseparation methods may facilitate one or more cell processing stepsincluding, but not limited to, cell concentration, cell dilution, cellwashing, buffer replacement, and magnetic separation. The cell selectionsystems described herein may increase throughput and cell yields output,in a compact and portable structure. For example, prior to magneticallyseparating cells, a suspension of cells may be mixed with magneticreagents in excess or at a predetermined concentration (e.g., cells/ml).Likewise, after magnetically separating cells, the cells may be washedin a solution (e.g., suitable buffered solution).

In some variations, a cell separation system may comprise a rotorconfigured for counterflow centrifugation elutriation of cells in afluid, a first magnet configured to magnetically rotate the rotor andseparate the cells from the fluid in the rotor, a flow cell in fluidcommunication with the rotor and configured to receive the cells fromthe rotor, and a second magnet configured to magnetically separate thecells in the flow cell.

In some variations, a CCE module may be integrated into a cartridge toenable a cell processing system to separate cells based on cell sizeand/or density. In some variations, a cell separation system maycomprise a housing comprising a rotor configured to separate cells froma fluid (e.g., separate cells of different size and/or density fromcells that remain in the fluid), and a magnet configured to magneticallyrotate the rotor. The housing may be configured to move relative to themagnet or vice versa (e.g., move the magnet relative to the housing).The CCE modules described herein may provide cell separation within acompact and portable housing where the magnet may be disposed externalto the housing (e.g., magnet disposed within a CCE instrument).

In some variations, a compact rotor that may aid cartridge integrationmay comprise input and output fluid conduits extending from the rotortowards opposing sides of a rotor housing. For example, a rotor maycomprise a first side comprising a first fluid conduit and a second sidecomprising a second fluid conduit where the second side is opposite thefirst side. An elutriation chamber (e.g., cone) may be coupled betweenthe first fluid conduit and the second fluid conduit.

In some variations, a method of separating cells from a fluid maycomprise moving a rotor towards a magnet, the rotor defining arotational axis, flowing the fluid through the rotor, rotating the rotor(e.g., magnetically) about the rotational axis using the magnet whileflowing the fluid through the rotor, and moving the rotor away from themagnet.

In some variations, a method of separating cells from a fluid maycomprise flowing the fluid comprising the cells into a flow cell. A setof the cells may be labeled with magnetic particles. The set of cellsmay be magnetically attracted towards a magnet array for a dwell time,and the set of cells may flow out of the flow cell after the dwell time.

In some variations, a flow cell may comprise an elongate cavity having acavity height and a magnet array comprising a plurality of magnets, eachof the magnets spaced apart by a spacing distance. A predetermined ratiobetween the cavity height to the spacing distance may optimize magneticseparation of the cells in the flow cell.

Electroporation

In some variations, an electroporation module as described herein may beconfigured to facilitate one or more of transduction and transfection ofcells. As described in more detail herein, a volume of fluid (e.g.,first batch) comprising cells may be physically separated from asubsequent volume of fluid (e.g., second batch, third batch) comprisingcells by a gas (e.g., air gap). Applying an electroporation signal(e.g., voltage pulse, waveform) separately to each discrete batch offluid may improve electroporation efficiency and thus increasethroughput. In some variations, active electric field compensation maysimilarly improve electroporation efficiency and throughput.

In some variations, a cell processor may comprise a fluid conduitconfigured to receive a first fluid comprising cells and a second fluid(e.g., gas, oil), a set of electrodes coupled to the fluid conduit, apump coupled to the fluid conduit, and a controller comprising aprocessor and memory. The controller may be configured to generate afirst signal to introduce the first fluid into the fluid conduit usingthe pump, generate a second signal to introduce the second fluid intothe fluid conduit such that the second fluid separates the first fluidfrom a third fluid, and generate an electroporation signal toelectroporate the cells in the fluid conduit using the set ofelectrodes.

In some variations, a method of electroporating cells may comprisereceiving a first fluid comprising cells in a fluid conduit, receiving asecond fluid comprising a gas in the fluid conduit to separate the firstfluid from a third fluid, and applying an electroporation signal to thefirst fluid to electroporate the cells.

In some variations, a method of electroporating cells may comprisereceiving a first fluid comprising cells in a fluid conduit, applying aresistance measurement signal to the first fluid using a set ofelectrodes, measuring a resistance between the first fluid and the setof electrodes, and applying an electroporation signal to the first fluidbased on the measured resistance.

Bioreactor

In some variations, a bioreactor may comprise an enclosure comprising abase and a sidewall, and a gas-permeable membrane coupled to one or moreof the base and the sidewall of the enclosure. The gas-permeablemembrane may aid cell culture. In some variations, a cell processingsystem may comprise the bioreactor and an agitator coupled to thebioreactor. The agitator may be configured to agitate the bioreactorbased on orbital motion.

Fluid Connector

Currently, there is no automated, multi-use sterile fluid connectorsolution for cell therapy production where a set of sterile fluidconnectors are capable of multiple connection and disconnection cycleswith a system. For example, conventional sterile fluid connectors aretypically single-use devices and are thus expensive and labor intensive.Generally, the fluid connectors described herein include a plurality ofsealed enclosures between a sterile portion (e.g., fluid connector lumenor cavity) and an external (e.g., non-sterile) ambient environment,thereby facilitating aseptic control of a fluid connector and devicescoupled thereto. The fluid connectors described herein may be a durablecomponent that may be reused for multiple cycles while maintainingsterility and/or bioburden control. For example, the fluid connector maybe sterilized using a sterilant without harming the cell product orother biological material.

In some variations, a sterile manufacturing system as described hereinmay utilize one or more sterile fluid connectors and have aconfiguration suitable to be manipulated by a robot such as a roboticarm. The sterile fluid connectors described herein enable the transferof fluids in an automated, sterile, and metered manner for automatingcell therapy manufacturing. Automating cell therapy manufacturing may inturn provide lower per patient manufacturing costs, a lower risk ofprocess failure, and the ability to meet commercial scale patient demandfor cell therapies. In some variations, sterile fluid connectors mayincrease one or more of sterility, efficiency, and speed by removing ahuman operator from the manufacturing process. An automated andintegrated sterilization process as described herein may be applied tothe fluid connector to maintain sterility of the system. For example,the fluid connector may maintain sterility through multipleconnection/disconnection cycles between separate sterile closed volumefluid devices (e.g., enclosure, container, vessel, cartridge,instrument, bioreactor, enclosed vessel, sealed chamber). Accordingly,the systems, devices, and methods described herein may reduce thecomplexity of a sterilization process, reduce energy usage, and increasesterilization efficiency.

In some variations, a fluid connector may comprise a first connectorconfigured to mate with a second connector (e.g., male connector andfemale connector). Respective proximal ends of the connectors may beconfigured to connect (e.g., be in fluid communication, form a fluidpathway) with respective fluid devices in order to transfer one or moreof fluid (e.g., liquid and/or gas) and biological material (e.g., cellproduct) between the fluid devices. The distal ends of the connectorsmay comprise ports configured to mate with each other. The fluidconnector may also comprise a sterilant port configured to facilitatesterilization of a chamber within the distal ends of the first andsecond connectors. The fluid connector may be sterilized before or afterconnection as desired to ensure sterility. In this manner, the fluidconnector may be reused for multiple connection and disconnectioncycles.

In some variations, a system (e.g., workcell) utilizing the fluidconnectors described herein may comprise a robot configured to operatethe fluid connector and a controller configured to control the robot tomanipulate (e.g., move, connect, open, close, disconnect) the first andsecond connectors together (without human interaction) while maintainingsterility of the fluid connector and a plurality of fluid devices,thereby further reducing the risk of contamination. The fluid devicesmay be one or more of an instrument, cartridge, and the like.

Cell Processing Control

Systems and methods for manufacturing cell products for biomedicalapplications using automated systems are described herein. Conventionalsemi-automated solutions for cell processing do not allow users todefine biological processes. Instead, users select from a limited set ofpredefined machine processes and process-control parameters. Currently,there is no scalable manufacturing solution for cell therapy production.For example, cell therapy manufacturing is conventionally executedbatchwise (i.e. one product will be manufactured in a single room/suite,with required processing tools located inside). This can either beguided by a technician following a standard operating procedure (SOP),or in some cases, processing tools (e.g., Miltenyi Prodigy, LonzaCocoon) can carry out a series of processing steps for a single patientproduct on a single multi-functional processing tool. However, existingsolutions (e.g., Miltenyi Prodigy) do not allow users to definebiological processes. Furthermore, the manual labor required ofconventional solutions increases the risk of product contamination andhuman error.

In some variations, a set of cell therapy biological manufacturingprocesses may be transformed into a set of machine instructions suitablefor automated execution using the systems described herein. For example,a method of transforming user-defined cell processing operations intocell processing steps to be executed by a processor of an automated cellprocessing system may comprise receiving an ordered input list of cellprocessing operations, and executing a transformation model on theordered input list to create an ordered output list of cell processingsteps capable of being performed by the system. As used herein, atransform model may refer to an algorithm, process, or transformationconfigured to translate a set of cell processing steps into a set ofmachine or hardware instructions for the system. In some variations, arobot may be controlled to move one or more cartridges each containing acell product between the instruments, and the instruments may becontrolled to perform cell processing steps on each cell product. Inthis manner, the systems and methods enable biologists to definemanufacturing processes in biological terms and have the systemtransform this biological model (e.g., process definition) into a set ofmachine-executed instructions.

The end-to-end closed system automation described herein may reduceprocess failure rates and cost. For example, end-to-end automation mayreduce manufacturing time (e.g., dwell times) and increase throughput ascompared to conventional manual methods. For example, a plurality ofprocesses (e.g., 10 or more) may be executed simultaneously. The methodsdescribed herein may further reduce opportunities for contamination anduser error. Thus, the systems, apparatuses, and methods described hereinmay increase one or more of cell processing automation, repeatability,reliability, process flexibility, instrument throughput, processscalability, and reduce one or more of labor costs, and processduration.

I. SYSTEM

Described here are systems and apparatuses configured to perform cellprocessing steps to manufacture a cell product (e.g., cell therapyproduct). In some variations, a cell processing system may comprise aplurality of instruments each independently configured to perform one ormore cell processing operations upon a cartridge (e.g., fluid device),and a robot capable of moving the cartridge between each of theplurality of instruments. The use of a robot and controller mayfacilitate one or more of automation, efficiency, and sterility of acell processing system.

In some variations, a system for cell processing may comprise aplurality of instruments each independently configured to perform one ormore cell processing operation upon a cartridge. A robot may be capableof moving the cartridge between each of the plurality of instruments. Insome variations, the system may be a workcell comprising an enclosure.

FIG. 1A is a block diagram of a cell processing system 100 comprising aworkcell 110 and controller 120. In some variations, the workcell 110may comprise one or more of an instrument 112, a cartridge 114 (e.g.,consumable, fluid device), a robot 116 (e.g., robotic arm), a reagentvault 118, a fluid connector 132, a sterilant source 134, a fluid source136, a pump 138, a sensor 140, and a sterile liquid transfer device 142.In some variations, the controller 120 may comprise one or more of aprocessor 122, a memory 124, a communication device 126, an input device128, and a display 130.

In some variations, a workcell may comprise a fully, or at leastpartially, enclosed housing inside which one or more cell processingsteps are performed in a fully, or at least partially, automatedprocess. In some variations, the workcell may be an open system lackingan enclosure, which may be configured for use in clean room, biosafetycabinet, or other sterile location. In some variations, the cartridge114 may be moved using the robot 116 to reduce manual labor in the cellprocessing steps. In some variations, the workcell may be configured toperform sterile liquid transfers into and out of the cartridge in afully or partially automated process. For example, one or more fluidsmay be stored in a sterile liquid transfer device 142. In somevariations, the sterile liquid transfer device may be a portableconsumable that may be moved within the system 100. The sterile liquidtransfer devices and fluid connectors described herein enable thetransfer of fluids in an automated, sterile, and metered manner forautomating cell therapy manufacturing. In some variations, the enclosureof the workcell may be configured to meet International Organization forStandardization (ISO) standard ISO7 or better (e.g., ISO6 or ISO5). Anadvantage of meeting ISO7 or better standards is that the system may beused in a facility that does not meet ISO7 standards (i.e. that lack aclean room or other sufficiently filtered air space). Optionally, thefacility may be an ISO8 or ISO9 facility. In some variations, a workcellmay comprise a volume of less than about 800 m³, less than about 700 m³,less than about 600 m³, less than about 500 m³, less than about 300 m³,less than about 250 m³, less than about 200 m³, less than about 150 m³,less than about 100 m³, less than about 50 m³, less than about 25 m³,less than about 10 m³, and less than about 5 m³, including all rangesand sub-values in-between.

In some variations, a robot 116 may be configured to manipulateconsumable cartridges 114 and fluid connectors 132 between differentinstruments to perform a predetermined sequence of cell processingsteps. In some variations, the same consumable cartridge 114 may bereceived by different instruments 112 and/or multiple cartridges 114 maybe processed in parallel.

In some variations, a cartridge 114 may contain cell product fromdifferent donors or contain cell product intended for differentrecipients. The cell product from a single donor may be split betweenmultiple cartridges 114 if necessary to generate enough product fortherapeutic use, or when a donor is providing product for severalrecipients (e.g., for allogeneic transplant). The cell product for asingle recipient may be split between multiple cartridges 114 ifnecessary to generate enough product for therapeutic use in thatrecipient. The cell product for a single recipient may be split betweenmultiple cartridges 114 if necessary to generate several cell productswith unique genetic modifications, and then optionally recombined incertain ratios for therapeutic use in that recipient. For example, afluid connector 132 may be coupled between two or more cartridges 114 totransfer a cell product and/or fluid between the cartridges 114.Furthermore, a fluid connector 132 may be coupled between any set offluid-carrying components of the system 100 (e.g., cartridge 114,reagent vault 118, fluid source 136, sterile liquid transfer device 142,fluid conduit, container, vessel, etc.). For example, a first fluidconnector may be coupled between a first cartridge and a sterile liquidtransfer device, and a second fluid connector may be coupled between thesterile liquid transfer device and a second cartridge.

As illustrated in FIG. 1B, a cartridge 114 may comprise one or more of abioreactor 150, cell separation system 152, electroporation module 160,liquid transfer bus 162, sensor 164, and fluid connector 166, asdescribed in more detail herein. A cell separation system 152 maycomprise one or more of a rotor 154, flow cell 156, and magnet 158. Insome variations, the magnet 158 may comprise one or more magnets and/ormagnet arrays. For example, the cell separation system 152 may comprisea first magnet configured to magnetically rotate a rotor 154 and asecond magnet (e.g., magnet array) configured to magnetically separatecells in flow cell 156.

Workcell

In some variations, a workcell 110 may comprise at least a partiallyenclosed enclosure (e.g., housing) in which one or more automated cellprocessing steps are performed. For example, the workcell 110 may beconfigured to transfer sterile liquid into and out of a cartridge 114 ina fully or partially automated process. In some variations, a workcell110 may not have an enclosure and be configured for use in a clean room,a biosafety cabinet, or other suitably clean or sterile location. Insome variations, the workcell 100 may comprise a feedthrough accessbiosafety cabinet, quality control instrumentation, pump, consumable(e.g., fluid device), fluid connector, consumable feedthrough, andsterilization system (e.g., sterilant source and/or generator, fluidsource, heater/dessicator, aerator).

FIG. 2A is a block diagram of a cell processing system including aworkcell 203. Workcell 203 may comprise an enclosure 202 having fourwalls, a base, and a roof. The workcell may be divided into an interiorzone 204 with a feedthrough 206 access, and quality control (QC)instrumentation 212. An air filtration inlet (not shown) may providehigh-efficiency particulate air (HEPA) filtration to provide ISO7 orbetter air quality in the interior zone 204. This air filtration maymaintain sterile cell processing in an ISO8 or ISO9 manufacturingenvironment. The workcell 203 may also have an air filter on the airoutlet to preserve the ISO rating of the room. In some variations, theworkcell 203 may further comprise, inside the interior zone 104, abioreactor instrument 214, a cell selection instrument 216 (e.g., MACS),an electroporation instrument (EP) 220, a counterflow centrifugationelutriation (CCE) instrument 222, a sterile liquid transfer instrument224 (e.g., fluid connector), a reagent vault 226, and a sterilizationsystem 260. The reagent vault 226 may be accessible by a user through asample pickup port 228. A robot 230 (e.g., support arm, robotic arm) maybe configured to move one or more cartridges 250 (e.g., consumables)from any instrument to any other instrument and/or move one or morecartridges 250 to and from a reagent vault. In some variations, theworkcell 203 may comprise one or more moveable barrier 213 (e.g.,access, door) configured to facilitate access to one or more of theinstruments in the workcell 203.

In some variations of methods according to the disclosure, a humanoperator may load one or more empty cartridges 250 into the feedthrough206 via cartridge port 207. The cartridges 250 may be pre-sterilized, orthe feedthrough 206 may sterilize the cartridge 250 using ultravioletradiation (UV), or chemical sterilizing agents provided as a vapor,spray, or wash. The feedthrough 206 chamber may optionally be configuredto automatically spray, wash, irradiate, or otherwise treat cartridges(e.g. with ethanol and/or isopropyl alcohol solutions, vaporizedhydrogen peroxide (VHP)) to maintain sterility of the interior zone 204(e.g., ISO 7 or better). The cartridge 250 may be passed to thebiosafety cabinet 206, where input cell product is provided and loadedto the cartridge through a sterile liquid transfer port into thecartridge 250. The user (via robot 230) may then move the cartridge 250back to the feedthrough 206 and initiate automated processing using acomputer processor in the computer server rack (e.g., controller 120).The robot 230 may be configured to move the cartridge 250 in apredefined sequence to a plurality of instruments and stations, with thecomponents of the workcell 200. At the end of cell processing, thecartridge 250, now containing the processed cell product, may bereturned to the feedthrough 206 for retrieval by the user. In somevariations, an outer surface of the enclosure 202 may comprise aninput/output device 208 (e.g., display, touchscreen).

FIG. 2B is a perspective view of a workcell 205 of a cell processingsystem. FIG. 2C is a perspective view of a cell processing systemdepicting a cartridge 250 (e.g., any of the cartridges described herein)introduced into a workcell 205 (e.g., any of the workcells describedherein). A plurality of cartridges may be inserted into the workcell 205simultaneously and undergo one or more cell processing operations inparallel.

In some variations, the workcell 205 may comprise a height of more thanabout a meter, between about 1 m and about 3 m, between about 1 m andabout 5 m, between about 3, and about 10 m, between about 5 m and about20 m, between about 10 m and about 30 m, between about 20 m and 100 m,and more than about 100 m, including all values and ranges in-between.In some variations, the workcell 205 may comprise one or more of alength and width of more than about 1 meter, between about 1 m and about5 m, between about 3, and about 10 m, between about 5 m and about 20 m,between about 10 m and about 30 m, between about 20 m and 100 m, andmore than about 100 m, including all values and ranges in-between.

FIG. 2D is a schematic illustration of a variation of a workcell 200.Workcell 200 may comprise an enclosure 202 having four walls, a base,and a roof. The workcell may be divided into an interior zone 204 with afeedthrough 206 access, a biosafety cabinet (BSC) 208, compute serverrack 210 (e.g., controller 120), and quality control (QC)instrumentation 212. An air filtration inlet (not shown) may providehigh-efficiency particulate air (HEPA) filtration to provide ISO7 orbetter air quality in the interior zone 204. This air filtration maymaintain sterile cell processing in an ISO8 or ISO9 manufacturingenvironment. The workcell may also have an air filter on the air outletto preserve the ISO rating of the room. In some variations, the workcell200 may further comprise, inside the interior zone 204, an instrument211 (e.g., disposed in a universal instrument bay), a bioreactorinstrument 214, a cell selection instrument 216 (e.g., MACS, cellselection system), a cell sorting instrument 218 (e.g., FACS), anelectroporation instrument (EP) 220, and a counterflow centrifugationelutriation (CCE) instrument 222, a sterile liquid transfer instrument224 (e.g., fluid connector), a reagent vault 226, and a sterilizationsystem 260 comprising one or more of a sterilant source, fluid source,and a pump. The reagent vault 226 may be accessible by a user through asample pickup port 228. A robot 230 (e.g., support arm, robotic arm) maybe configured to move one or more cartridges 250 (e.g., consumables)from any instrument to any other instrument or reagent vault.

In some variations, a human operator may load one or more cartridges 250into the feedthrough 206. The cartridges 250 may be pre-sterilized, orthe feedthrough 206 may sterilize the cartridge 250 using ultravioletradiation (UV), or chemical sterilizing agents provided as a spray orwash. The feedthrough 206 chamber may optionally be configured toautomatically spray, wash, irradiate, or otherwise treat cartridges(e.g. with ethanol and/or isopropyl alcohol solutions) to maintainsterility of the interior zone 204 (e.g., ISO 7 or better) or thebiosafety cabinet 208 (e.g., ISO 5 or better). The cartridge 250 may bepassed to the biosafety cabinet 206, where input cell product isprovided and loaded to the cartridge using a sterile liquid transferinstrument 224 (e.g., fluid connector) into the cartridge 250. The usermay then move the cartridge 250 back to the feedthrough 206 and initiateautomated processing using a computer processor in the computer serverrack 210 (e.g., controller 120). The robot 230 may be configured to movethe cartridge 250 in a predefined sequence to a plurality of instrumentsand stations, with the components of the workcell 200 being controlledby the computer processor of the computer server rack 210. Additionallyor alternatively, the sequence that the cartridge 250 moves within theworkcell 200 may not be predefined. For example, cartridge 250 movementmay not be dependent on one or more of the result of a previous step,sensor value, predetermined threshold (e.g., based on a quality controlsystem), and the like. At the end of cell processing, the cartridge 250,now containing the processed cell product, may be returned to thefeedthrough 206 for retrieval by the user. Additionally oralternatively, the cell product 250 containing the processed cellproduct may be transferred (via a fluid connector) to a second cartridge(e.g., single-use cartridge) and stored in the reagent vault 226 forretrieval by the user.

In some variations, cells from a patient and starting reagents may beloaded into a cartridge (e.g., single-use cartridge) by a human operatorin a biosafety cabinet located separate from the workcell or integratedinto the workcell. In some variations, the cartridges described hereincomprising a cell product and reagent may move through a non-sterilefield without contamination since the cartridge is closed. The cartridgemay further undergo an automated decontamination routine. For example,the cartridge may be placed within a feedthrough capable of facilitatingdecontamination of the cartridge before entering the ISO 7 environmentin the workcell.

FIG. 2E is a plan schematic illustration of another variation of aworkcell 201. Workcells 200, 201, and 203 may comprise an enclosure 202having four walls, a base, and a roof. The workcell may be divided intoan interior zone 204 with a feedthrough 206 access, a biosafety cabinet(BSC) 208, compute server rack 210 (e.g., controller 120), and qualitycontrol (QC) instrumentation 212. An air filtration inlet (not shown)may provide high-efficiency particulate air (HEPA) filtration to provideISO7 or better air quality in the interior zone 204. This air filtrationmay maintain sterile cell processing in an ISO8 or ISO9 manufacturingenvironment. The workcell may also have an air filter on the air outletto preserve the ISO rating of the room. In some variations, the workcell200 may further comprise, inside the interior zone 104, an instrument211 (e.g., disposed in a universal instrument bay), a bioreactorinstrument 214, a cell selection instrument 216 (e.g., MACS), a cellsorting instrument 218 (e.g., FACS), an electroporation instrument (EP)220, and a counterflow centrifugation elutriation (CCE) instrument 222,a sterile liquid transfer instrument 224, and a reagent vault 226. Thereagent vault 226 may be accessible by a user through a sample pickupport 228 (e.g., a door which may facilitate bulk loading of sterileliquid transfer instruments 224). A robot 230 (e.g., support arm,robotic arm) may be configured to move one or more cartridges 250 (e.g.,consumables) from any instrument to any other instrument or reagentvault.

In some variations of methods according to the disclosure, a humanoperator may load one or more empty cartridges 250 into the feedthrough206. Additionally or alternatively, pre-filled cartridges may be loadedinto the feedthrough 206. The cartridges 250 may be pre-sterilized, orthe feedthrough 206 may sterilize the cartridge 250 using ultravioletradiation (UV), or chemical sterilizing agents provided as a spray orwash. The feedthrough 206 chamber may optionally be configured toautomatically spray, wash, irradiate, or otherwise treat cartridges(e.g. with ethanol and/or isopropyl alcohol solutions) to maintainsterility of the interior zone 204 (e.g., ISO 7 or better) or thebiosafety cabinet 208 (e.g., ISO 5 or better). The cartridge 250 may bepassed to the biosafety cabinet 106, where input cell product isprovided and loaded to the cartridge through a sterile liquid transferport into the cartridge 250. The user may then move the cartridge 250back to the feedthrough 206 and initiate automated processing using acomputer processor in the computer server rack 210 (e.g., controller120). The robot 230 may be configured to move the cartridge 250 in apredefined sequence to a plurality of instruments and stations, with thecomponents of the workcell 200 being controlled by the computerprocessor of the computer server rack 210. At the end of cellprocessing, the cartridge 250, now containing the processed cellproduct, may be returned to the feedthrough 206 for retrieval by theuser.

In some variations, one or more components of a sterilization system(e.g., sterilant source, pump) may be coupled to a workcell. Forexample, FIG. 3 is a block diagram of a cell processing system 300comprising a workcell 310, sterilization system 320, fluid connector 330and fluid devices 340. In some variations, the fluid devices 340 maycomprise a main (e.g., consumable) feedthrough and a fluid device (e.g.,reagent) feedthrough. The sterilization system 320 may comprise asterilant source 322, pump 324, and heater (e.g., desiccant/dryer) 326.For example, the heater 326 may be configured to aerate at apredetermined set of conditions. The sterilization system 320 may becoupled and in fluid communication with one or more of the workcell 310,fluid connector 330, and fluid device 340. In some variations, a robot(not shown) may be configured to manipulate and operate the cellprocessing system 300. For example, the fluid connector 330 may becoupled to one or more of the fluid devices 340 and instruments (notshown). One or more of the workcell 310, fluid connector 330, and fluiddevices 340 may be sterilized and/or aerated by circulating one or moreof a sterilant and fluid (e.g., heated air, vaporized hydrogen peroxide(VHP)) using the sterilization system 320. In some variations, thesterilization system 320 may comprise one or more of vaporized hydrogenperoxide (VHP), electron-beam (e-beam) sterilization, dry thermaldecontamination, and steam-in-place. In some variations, thesterilization system 320 may provide a sterility assurance level (SAL)of at least 10-3 SAL.

FIGS. 4A and 4B illustrate perspective views of a cell processing system400 comprising a cartridge 400, 402, feedthrough 410, 412, and fluidconnector 420, 422 (e.g., sterile liquid transfer instrument). Forexample, cartridge 400 is shown in the feedthrough 410 in FIG. 16A whilea robot (not shown) has moved cartridge 400 to fluid connector 420.

Robot

Generally, a robot may comprise any mechanical device capable of movinga cartridge from one location to another location. For example, therobot may comprise a mechanical manipulator (e.g., an arm) in a fixedlocation, or attached to a linear rail, or a 2- or 3-dimensional railsystem. In a variation, the robot comprises a robotic shuffle system. Ina further variation, the robot comprises a wheeled device. In somevariations, the system comprises two or more robots of the same ordifferent type (e.g., two robotic arms each independently configured formoving cartridges between instruments). The robot may also comprise anend effector for precise handling of different cartridges or barcodescanning or radio-frequency identification tag (RFID) reading.

FIG. 5 is a perspective view of a cell processing system 500 in which arobot arm moves consumable cartridges between slots in variousinstruments each configured to perform a different cell processing step.In some variations, the same consumable cartridge can be received bydifferent instruments. The system 500 may comprise a modular design toaccommodate different instrument configurations. In some variations, aplurality of cartridges may be processed in parallel. Each cartridge maycontain a cell product from different donors or contain a cell productintended for different recipients. For example, a cell product from asingle donor may be split between a plurality of cartridges to generatea predetermined quantity of cell product for therapeutic use such aswhen a donor is providing product for several recipients (e.g., forallogeneic transplant). In some variations, the cell product for asingle recipient may be split between a plurality of cartridges togenerate a predetermined quantity of product for therapeutic use in thatrecipient. In some variations, the cell product for a single recipientmay be split between a plurality of cartridges to generate apredetermined quantity of several cell products with unique geneticmodifications, which may be recombined in certain ratios for therapeuticuse in that recipient.

Cartridge

Generally, the cell processing systems described herein may comprise oneor more cartridges including one or more modules configured to interfacewith an instrument or instruments. A robot (e.g., robotic arm) may beconfigured to move a cartridge and/or instrument to perform one or morecell processing steps. For example, a cartridge may comprise abioreactor module and/or fluid connector (e.g., sterile liquid transferport) coupled by the robot to a bioreactor instrument of a workcell.Once a predetermined processing step has been completed, the cartridgemay be moved by the robot to another instrument of the workcell, andanother cartridge may be coupled to the bioreactor instrument. Thus, aportable cartridge and shareable instruments may increase theefficiency, throughput, and flexibility of a cell manufacturing process.

In some variations, the cartridge may optionally provide aself-contained device capable of performing one or more cell processingsteps. The modules may be integrated into a fixed configuration withinthe cartridge. Additionally or alternatively, the modules may beconfigurable or moveable within the cartridge, permitting variouscartridges to be assembled from shared modules. Similarly stated, thecartridge can be a single, closed unit with fixed components for eachmodule; or the cartridge may contain configurable modules coupled byconfigurable fluidic, mechanical, optical, and electrical connections.In some variations, one or more sub-cartridges, each containing a set ofmodules, may be configured to be assembled to perform various cellprocessing workflows. The modules may each be provided in a distincthousing or may be integrated into a cartridge or sub-cartridge withother modules. The disclosure generally shows modules as distinct groupsof components for the sake of simplicity, but may be arranged in anysuitable configuration. For example, the components for differentmodules may be interspersed with each other such that each module isdefined by the set of connected components that collectively perform apredetermined function. However, the components of each module may ormay not be physically grouped within the cartridge. In some variations,multiple cartridges may be used to process a single cell product throughtransfer of the cell product from one cartridge to another cartridge ofthe same or different type and/or by splitting cell product into morecartridges and/or pooling multiple cell products into fewer cartridges.

Generally, each of the instruments of the system interfaces with itsrespective module or modules on the cartridge e.g., an electroporationmodule on the cartridge (if present) is moved by the system to anelectroporation instrument and interfaces with the electroporationinstrument to perform an electroporation step on the cell product—andmay also interface with common components, such as components of afluidic bus line (e.g., pumps, valves, sensors, etc.). An advantage ofsuch split module/instrument designs is that expensive components (e.g.,motors, sensors, heaters, lasers, etc.) may be retained in theinstruments of the system while multiple cartridges are processed. Theuse of disposable cartridges may eliminate the need, in such variations,to sterilize cartridges between use. Furthermore, the utilization ofshared instruments (e.g. electroporation instrument, CCE instrument,MACS instrument, sterile liquid transfer instrument, FACS instrument,and the like) may be increased since a plurality of the instruments maybe utilized simultaneously in parallel by a plurality of cellmanufacturing processes. In contrast, conventional semi-automatedinstruments (e.g., Miltenyi Prodigy) have instrument components that sitidle and are incapable of simultaneous parallel use.

FIG. 6 is a schematic illustration of a cartridge 600 that may be aconsumable produced from materials at a cost that make recycling orlimited use practical. The cartridge 600 may comprise a liquid transferbus 624 fluidically coupled to a small bioreactor module 614 a, a largebioreactor module 614 b, a cell selection module 616, a cell sortingmodule 618, an electroporation module 620, and a counterflowcentrifugation elutriation (CCE) module 622. In some variations, thecell selection module 616 may be a magnetic-activated cell selection(MACS) module. The cell sorting module 618 may comprise a fluorescenceactivated cell sorting (FACS) module. The cartridge 600 may comprise ahousing 602 that renders the cartridge self-contained, and optionallyprotects the contents from contamination. Sterile liquid transfer ports(SLTPs) 606 a-606 k may be fluidically coupled to reservoirs 607 a-607k, and each independently be a flexible bag or a rigid container. Insome variations, flexible bags may be configured to hold large volumesand to permit transfer of fluid without replacing transferred fluid withliquid or gas to maintain the pressure in the reservoir, as the bag maycollapse when fluid is transferred out and expand when fluid istransferred in.

In some variations, the liquid transfer bus 624 may comprise valves V1to V28 and corresponding tubing that fluidically links the valves to oneanother and to each of the modules. Valves shown coupled to four fluidiclines are 4/2 (4 port 2 position) valves and valves shown coupled tothree fluidic lines are 3/2 (3 port 2 position) valves. Internal flowpaths of the valves are indicated in the legend. The cartridge mayfurther comprise a first pump 632 a and a second pump 632 b, each ofwhich expose tubing on the exterior of the housing 602 to permit eachpump to interface with pump actuators (e.g., rotors) in some instrumentsin the system (e.g., workcell). The liquid transfer bus 624 may befluidically coupled to reservoir 607 d and a product bag which isfluidically coupled to STLP 606 d and to product input tubing lines 627a-627 b. An operator may input a cell product into reservoir 607 d byconnecting product input tubing line 627 a or 627 b to an externalsource of cells (e.g., a bag of cells collected from a donor). SLTP 606d may be configured to permit a system according to the disclosure(e.g., workcell 110) to add fluid to the reservoir 607 d in an automatedfashion. For example, one or more fluid-carrying containers such asreservoirs 607 a-607 k, bags, etc. may receive fluid using an SLTP.Additionally or alternatively, the SLTP may be configured toperiodically sample one or more of the fluid-carrying containers. Thecartridge may further comprise collection bags 626 a-626 c, fluidicallycoupled to the liquid transfer bus 624 via valves V17-V19. The cartridge600 may be configured to permit an operator to remove the collectionbags 626 a-626 c after completion of cell processing by the system.

FIG. 7 is a schematic diagram of another variation of a cartridge 700.For example, cartridge 700 may comprise a reduced feature set comparedto cartridge 600. The cartridge 700 may comprise a liquid transfer bus724 fluidically coupled to a bioreactor module 714, a counterflowcentrifugation elutriation (CCE) module 722, and a module 716 selectedfrom cell selection module, a cell sorting module, an electroporationmodule, or any other cell processing module. The cartridge 700 maycomprise a housing 702 and sterile liquid transfer ports (SLTPs) 706a-706 f (e.g., fluid connector) fluidically coupled to reservoirs 707a-707 f, which may be each independently be a flexible bag or a rigidcontainer. SLTP 706 g is fluidically coupled to the bioreactor module714 to permit direct access by a system or an operator to thebioreactor. Reservoir 707 c may be fluidically coupled to SLTP 707 c andproduct input tubing line 727. In some variations, the liquid transferbus 724 may comprise 14 valves V1-V3, V9, V11-V12, V17-V23 and V28 andtubing that fluidically couples the values to one another and/or each ofthe modules. The cartridge may further comprise collection bags 726a-726 c fluidically coupled to the liquid transfer bus 724 via valvesV17-V19. The cartridge may further comprise a pump 732 which exposes thetubing on the exterior of the housing 702 to permit each pump tointerface with a pump actuator in the system (e.g., workcell).

A side and top view of another variation of a cartridge is shown inrespective FIGS. 8A and 8B. In some variations, a cartridge 800 maycomprise a bioreactor 814, a pump 816, and a counterflow centrifugationelutriation (CCE) module 822. The cartridge 800 may comprise blanks 818,819, and 820 configured to house additional module(s) such as a cellselection module, cell sorting module, an electroporation module, asmall bioreactor module, and the like. In some variations, a blank maydefine an empty volume of the cartridge reserved to house a module atanother time. In some variations, the cartridge 800 may comprise two ormore additional bioreactors and/or reservoirs in blanks 818, 819, 820.Along the near surface of the cartridge 800 may be fluid connectors 806a-806 j (e.g., SLTP) fluidically connected to reservoirs 807 a-807 f.Reservoirs 807 b and 807 e may comprise fluid (e.g., buffer or media).Along the top surface are product input tubing lines 827 a-827 d, whichmay be fluidically connected to reservoirs 807 a, 807 b, 807 e, and 807f, respectively. A liquid transfer bus 824 may fluidically connect theSTLPs, reservoirs, and product input tubing lines to the modules viatubing.

In some variations, the housing 802 may have external dimensions ofabout 225 mm×about 280 mm×385 mm, about 225 mm×about 295 mm×385 mm, andabout 450 mm×about 300 mm×about 250 mm, including all values andsub-ranges in-between. In some variations, the cartridge 800 may beabout 10%, about 20%, about 30% or more smaller in volume, including allranges and sub-values in-between. In some variations, the cartridge 800may be about 10%, about 20%, about 30%, about 50%, about 100%, about200%, or more in volume, including all ranges and sub-values in-between.

In some variations, a cartridge 800 as shown in the side view of FIG. 8Cand perspective view of FIG. 8D may comprise a MACS module 818. Forexample, the bioreactor module 814 may comprise ports 815 a-815 fincluding a pH and dissolved oxygen (DO) sensors (ports 815 a and 815b), a gas input line 815 c, an output line 815 d each having a sterilefilter behind the connector, and a coolant input line 815 e and outputline 815 f from the bioreactor instrument interface when it interfaceswith bioreactor module 814 (for heat exchange). For example, the gasinput line 815 c may be configured for gas transfer into a fluid (e.g.,through headspace gas control or a gas-permeable membrane).

FIG. 9 shows a cross-sectional side view of a cartridge 900. In somevariations, a cartridge 900 may comprise an enclosure (e.g., housing), abioreactor 914, one or more pumps 916, valve 930, cell selection module917, and a counterflow centrifugation elutriation (CCE) module 922. Insome variations, the cell selection module 616 may be amagnetic-activated cell selection (MACS) module 917. The cartridge mayfurther comprise collection bags 926. The cartridge 900 may optionallycomprise blanks configured to house additional module(s) such as a cellselection module, a cell sorting module, an electroporation module 918,and the like. In some variations, the cartridge 900 may comprise one ormore bioreactors and/or reservoirs in the blanks.

In some variations, a cartridge may comprise one or more valves. In somevariations, the valve 1000 on the cartridge may be configured to receivean actuator 1010 provided by an instrument (as shown in FIG. 10A). Asthe cartridge is inserted into the instrument, the valve 1000 may beconfigured to dock with the actuator 1010 (as shown in FIG. 10B), suchthat rotation of the actuator 1010 may cause switching of the valve 1000from one position to another position. In some variations, the valvesmay be constructed to pinch a section of soft tubing. The pinch valvesmay comprise a closed configuration, and an external actuator may beconfigured to interface with the pinch valve (e.g., utilizing a solenoidwith linear motion) to open or close the valve. The valves themselvesmay be configured to be disposable whereas the actuators may beintegrated into an instrument configured to process cartridgesrepeatedly.

Reagent Vault

In some variations, the system comprises a reagent vault (or reagentvaults) where reagents are stored including but not limited to cellculture media, buffer, cytokines, proteins, enzymes, polynucleotides,transfection reagents, non-viral vectors, viral vectors, antibiotics,nutrients, cryoprotectants, solvents, cellular materials, andpharmaceutically acceptable excipients. Additionally or alternatively,waste may be stored in the reagent vault. In some variations, in-processsamples extracted from one or more cartridges may be stored in thereagent vault. The reagent vault may comprise one or more controlledtemperature compartments (e.g., freezers, coolers, water baths, warmingchambers, or others, at e.g. about −80° C., about −20° C., about 4° C.,about 25° C., about 30° C., about 37° C., and about 42° C.).Temperatures in these compartments may be varied during the cellmanufacturing process to heat or cool reagents. In variations of themethods of the disclosure, a cartridge may be moved by the robot (ormanually by an operator) to the reagent vault. The reagent vaultinterfaces with one or more sterile liquid transfer ports on thecartridge, and the reagent or material is dispensed into the cartridge.Optionally, fluid is added or removed from the cartridge before, during,or after reagent addition or removal. In some variations, the systemcomprises a sterile liquid transfer instrument, similarly configured totransfer fluid into or out of the cartridge in an automated, manual, orsemi-automated fashion. An operator may stock the sterile liquidtransfer station with reagents manually, or they may be supplied by arobot (e.g. from a feedthrough or other location). In some cases, arobot moves a reagent or reagents from the reagent vault to the sterileliquid transfer station. The reagent vault may have automated doors topermit access by the robot for sterile liquid transfer devices and/orother reagent vessels, optionally each under independent closed looptemperature control. The devices and vessels may be configured forpick-and-place movement by the robot. In some variations, the reagentvault may comprise one or more sample pickup areas. For example, a robotmay be configured to move one or more reagents to and from one or moreof the sample pickup areas.

Various materials can be used to construct the cartridge and thecartridge housing, including metal, plastic, rubber, and/or glass, orcombinations thereof. The cartridge, its components, and its housing maybe molded, machined, extruded, 3D printed, or any combination thereof.The cartridge may contain components that are commercially available(e.g., tubing, valves, fittings); these components may be attached orintegrated with custom components or devices. The housing of thecartridge may constitute an additional layer of enclosure that furtherprotects the sterility of the cell product. The operator may performloading or unloading of the cartridge in an ISO 5 or better environment,utilizing aseptic technique to ensure that sterility of the contents ofthe cartridge is maintained when the cartridge is opened. In somevariations, the operator may perform loading or unloading of thecartridge using manual aseptic connections (e.g., sterile tube welding).The robotic system may also perform sterile loading or unloading ofliquids into and out of the cartridge through the use of the sterileliquid transfer instrument and sterile liquid transfer ports on thecartridge.

Counterflow Centrifugal Elutriation

Counterflow centrifugal elutriation (CCE) is a technique used toseparate cells based on characteristics such as size and/or density.Counterflow centrifugal elutriation combines centrifugation withcounterflow elutriation where centrifugation corresponds to the processof sedimentation under the influence of a centrifugal force field andcounterflow elutriation corresponds to the process of separation bywashing. Separation takes place in a cone (e.g., bicone, funnel) shapedelutriation chamber. Particles (e.g., cells) conveyed in a fluid intothe elutriation chamber are acted upon by two opposing forces:centrifugal force driving the fluid away from an axis of rotation; andfluid velocity driving the fluid towards the axis of rotation (e.g.,counterflow). By varying the flow rate and the centrifugal force, theseparation of particles (e.g., cells) may be achieved. For example, asdescribed in more detail herein, particles may be separated based onproperties such as size and density.

Counterflow centrifugal elutriation may perform multiple operationsuseful for cell therapy manufacturing workflows including, but notlimited to, cell washing, cell concentration, media/buffer replacement,transduction, and separation of white blood cells from other bloodcomponents (e.g., platelets, and red blood cells). In some variations, afluid source (e.g., apheresis bag) for a cell separation process maycomprise a suspension of white blood cells, red blood cells, platelets,and plasma. In order to separate immune cells of interest, white bloodcells may be isolated and subsequently magnetically tagged for magneticseparation. A white blood cell separation step may be performed in a CCEmodule to separate cells based on size and density, while magneticseparation may be performed in a MACS module. In some variations, a CCEmodule may be integrated into a cartridge to enable a cell processingsystem to separate cells based on one or more of a progression through acell cycle (e.g., G₁/M phase cells being larger than G₀, S, or G2 phasecells) and cell type (e.g., white blood cells from red blood cellsand/or platelets).

Generally, a rotor configured to spin may comprise an elutriationchamber (e.g., cone, bicone). A fluid comprising a suspension of cellsmay be pumped under continuous flow into the rotor. As cells areintroduced into the cone (e.g., bicone), the cells migrate according totheir sedimentation rates to positions in the gradient where the effectsof the two forces upon them are balanced. Smaller cells having lowsedimentation rates (e.g., platelets) may be quickly washed toward theaxis of rotation with increased flow velocity. Such smaller cells may beoutput (e.g., washed out) of the cone. Relatively larger (or denser)cells (e.g., red blood cells) flow through the cone relatively moreslowly and reach equilibrium at an elutriation boundary where thecentrifugal force and the drag force are in balance, and the fluidvelocity is relatively low because the cone has widened. The largest ordensest cells (e.g., white blood cells) remain near the inlet to thechamber where centrifugal force and fluid velocity are high. Byincreasing the flow rate in gradual steps, successive fractions ofincreasingly large or dense cells (e.g., platelets→red blood cells→whiteblood cells) may be output from the rotor. Continued incrementalincreases in fluid flow rate will eventually elutriate all cells fromthe cone.

FIG. 56 is a block diagram of a cell separation system 5600 comprising aworkcell 5610 and at least one cartridge 5620. In some variations, theworkcell 5610 may comprise one or more of a counterflow centrifugalelutriation (CCE) instrument 5632 (e.g., first magnet), amagnetic-activated cell selection (MACS) instrument 5642 (e.g., magnetarray, second magnet), a fluid connector 5652, a pump 5654, an imagingsystem comprising an optical sensor 5660 and an illumination source5662, a sensor 5664, and a processor 5670. In some variations, thecartridge 5620 may comprise one or more of a CCE module 5630 (e.g.,rotor), a MACS module 5640 (e.g., flow cell), and a fluid connector 5650(e.g., sterile liquid transfer port, liquid transfer bus). For example,a cartridge for cell processing may comprise a liquid transfer bus and aplurality of modules, each module fluidically linked to the liquidtransfer bus. The modules may include any of the CCE modules or MACSmodules described herein. In some variations, a robot (not shown) may beconfigured to move the cartridge 5620 between different locations withinthe workcell 5610 to perform different cell processing steps.

In some variations, the imaging system (e.g., optical sensor 5660,illumination source 5622) may be configured to generate image datacorresponding to one or more of the CCE module 5630 and MACS module5640. For example, image data of fluid flow through a rotor of a CCEmodule 5630 may be analyzed and used to control a flow rate of fluidand/or rotation rate of the rotor, as described in more detail herein.In some variations, the optical sensor 5660 may be a CMOS/CCD sensorhaving, for example a resolution of about 100 μm, a working distance ofbetween about 40 mm and about 100 mm, and a focal length of less thanabout 8 mm. The optical sensor 5660 may be configured to operatesynchronously with the illumination source 5662. In some variations, theoptical sensor 5660 may comprise one or more of a colorimeter, turbiditysensor, and optical density sensor. In some variations, the illuminationsource 5662 may operate as a strobe light configured to output lightpulses synchronized to a rotation rate of a rotor of the CCE module5630.

In some variations, the sensor 5664 may comprise one or more of anoptical density sensor configured to measure an intensity of fluid, aleak detector configured to detect moisture and/or leaks, an inertialsensor configured to measure vibration, a pressure sensor configured tomeasure pressure in a fluidic line (e.g., photoelectric sensor), abubble sensor configured to detect the presence of a bubble in a fluidconduit, colorimetric sensor, vibration sensor, and the like.

In some variations, the fluid connector 5652 may comprise one or morevalves, configured to control fluid flow between the workcell and thecartridge 5620. The processor 5670 may correspond to the controller(e.g., processor and memory) described in more detail herein. Theprocessor 5670 may be configured to control one or more of the CCEinstrument 5632, the MACS instrument 5642, the pump 5654, fluidconnector 5652 (e.g., valves), the optical sensor 5660, the illuminationsource 5662, and the sensors 5664.

In some variations, a system 5600 for cell processing may comprise acartridge 5600 comprising a rotor of a CCE module 5630 configured forcounterflow centrifugation elutriation of cells in a fluid. A firstmagnet of a CCE instrument 5632 may be configured to magnetically rotatethe rotor and separate the cells from the fluid in the rotor. Thecartridge may further comprise a flow cell of a MACS module 5640 coupledto the rotor and configured to receive the cells from the rotor. Asecond magnet of a MACS instrument 5642 may be configured tomagnetically separate the cells in the flow cell.

In some variations, an illumination source 5662 may be configured toilluminate the cells. An optical sensor 5660 may be configured togenerate image data corresponding to the cells. In some variations, thesystem 5600 may comprise one or more of an oxygen depletion sensor, leaksensor, inertial sensor, pressure sensor, and bubble sensor. In somevariations, the system 5600 may comprise one or more valves and pumps.

FIG. 57 is a cross-sectional side view of a counterflow centrifugalelutriation (CCE) module 5700 comprising a housing 5710 (e.g.,enclosure), a rotor 5720 configured to rotate within and relative to thehousing 5710, and one or more fluid ports 5730 (e.g., fluid inlet, fluidoutlet). In some variations, the CCE module 5700 may be portable andconfigured to move within a workcell 5610 and cartridge 5620. Forexample, a robot may move the CCE module 5700 between differentinstruments of a workcell 5610.

FIG. 58 is a cross-sectional side view of a magnetic-activated cellselection (MACS) module comprising a housing 5810 (e.g., enclosure), afirst fluid port 5820 (e.g., fluid inlet), a second fluid port 5830(e.g., fluid outlet), and a flow cell 5810 coupled in between the firstfluid port 5820 and the second fluid port 5830. As described in moredetail herein, the flow cell 5810 may comprise a cavity (e.g., chamber)comprising one or more channels (e.g., linear channels, laminar fluidflow channel). In some variations, the cavity of the flow cell 5810 maybe substantially empty. For example, the flow cell 5810 may be absent amesh, beads, tortuous channels, and the like. In some variations, theflow cell 5810 may have a longitudinal axis aligned perpendicular toground. That is, the flow cell 5810 may be oriented vertically where thefirst fluid port 5820 is disposed at a higher elevation than the secondfluid port 5830 such that gravity may aid fluid flow through the flowcell 5810. In some variations, the MACS module 5800 may be portable andconfigured to move within a workcell 5610 and cartridge 5620. Forexample, a robot may move the MACS module 5630 between differentinstruments of a workcell 5610.

FIGS. 59A and 59B are perspective views of a system 5900 for cellprocessing (e.g., CCE system) comprising a CCE module 5930 (e.g.,cartridge) including a housing 5931 and a rotor 5910, a CCE instrument5932, an optical sensor 5960, and an illumination source 5962. In somevariations, the CCE instrument 5932 may comprise a magnet configured tomagnetically rotate the rotor 5910 within the CCE module 5930. One ormore portions of the housing 5931 and rotor 5910 may be opticallytransparent to facilitate illumination by the illumination source 5962and image data generation by the optical sensor 5960.

In some variations, the system 5900 for cell processing may comprise acartridge 5930 comprising a housing 5931 comprising a rotor 5910configured to separate cells from a fluid. An instrument 5932 comprisinga magnet may be configured to interface with the cartridge 5930 tomagnetically rotate the rotor 5910. The cartridge 5930 may be configuredto move a cell product between a plurality of instruments. In somevariations, the housing 5931 may enclose the rotor 5910. In somevariations, the housing 5931 may comprise one or more apertures 5937configured to facilitate visualization (e.g., imaging) of the rotor5910. FIGS. 59A and 59B depict a magnet 5932 in proximity, but notattached, to housing 5931. FIG. 59C is a perspective view of the rotor5910 and housing 5931 without the magnet 5932, optical sensor 5960, andillumination source 5962.

In some variations, the cartridge 5930 (e.g., housing 5931, 5910) maycomprise a consumable component such as a disposable component, limiteduse component, single use component, and the like. In some variations,the magnet 5932 may comprise a durable component that may be re-used aplurality of times. In some variations, the magnet 5932 may bereleasably coupled to the housing 5931. For example, the housing 5931may be moved relative to the magnet 5932 to facilitate magnetic couplingbetween the magnet 5932 and a plurality of cartridges 5930. Additionallyor alternatively, the magnet 5932 may be configured to be moved relativeto the housing 5931.

FIG. 59D is a side cross-sectional view of a CCE module 5930. In somevariations, the housing 5931 of the rotor 5910 may comprise a first side5933 comprising the first fluid port 5912 (e.g., first fluid conduit)and a second side 5935 comprising the second fluid port 5914 where thesecond side 5935 is opposite the first side 5933. The rotor 5910(including a cone or bicone as described in more detail herein) may becoupled between the first fluid port 5912 and the second fluid port5914. In some variations, the CCE module 5930 may comprise an air gap5902 between the housing 5931 and a magnet 5932. That is, the cartridge5930 and magnet 5932 may couple in a non-contact manner. Consequently,the cartridge need not mechanically couple to the magnet 5932 to performcounterflow centrifugal elutriation. Therefore, the rotor 5910 may havea low alignment sensitivity with the magnet 5932, as well as lowvibration between the rotor 5910 and the magnet 5932. Furthermore, thespace between the rotor 5910 and magnet 5932 enables the second fluidport 5914 to extend toward the second side 5935 of the housing 5931,thus allowing for fluid to flow on each side of the rotor 5910.

In some variations, counterflow centrifugal elutriation may be performedby the system 5900 by moving a magnet 5932 towards a rotor 5910 (or viceversa). The rotor may define a rotational axis (e.g., coaxial with thefirst fluid port 5912 and the second fluid port 5914). Fluid may flowthrough the rotor via the first fluid port 5912 and the second fluidport 5914. The magnet 5932 may magnetically rotate the rotor about therotational axis while flowing the fluid through the rotor 5910. Therotor may move away from the magnet. For example, moving the rotor 5910may include advancing and withdrawing the rotor 5910 relative to themagnet 5932 using a robot (not shown).

In some variations, fluid may flow through first fluid port 5912 alongthe first side 5933 of the rotor 5910 and into the rotor 5910. Aftercounterflow centrifugal elutriation through the rotor 5910, the fluidmay flow out of the rotor 5910 through second fluid port 5914 along thesecond side 5935 of the rotor 5910.

In some variations, counterflow centrifugal elutriation may bevisualized by optical sensor 5960 and illumination source 5962 in orderto monitor and modify cell separation in real-time based onpredetermined criteria in a closed loop manner in order to maximizeelutriation efficiency. In some variations, an optical sensor 5960 maybe configured to image any portion of the rotor through which fluidflows (e.g., first fluid conduit, second fluid conduit, third fluidconduit, first bicone, second bicone). For example, image data of one ormore of the fluid and the cells in the rotor 5910 may be generated usingthe optical sensor 5960. In some variations, one or more of the fluidand the cells may be illuminated using the illumination source 5962. Forexample, an output of a cone may be imaged by an optical sensor toidentify non-target cells being elutriated.

In some variations, one or more of a rotation rate of the rotor and aflow rate of the fluid may be selected based at least in part on theimage data. For example, the rotor may comprise a rotation rate of up to6,000 RPM. For example, the fluid may comprise a flow rate of up toabout 150 ml/min while rotating the rotor. In some variations, the rotormay be moved towards the illumination source 5962 and the optical sensor5960. Additionally or alternatively, the rotor 5910 may be moved awayfrom the illumination source 5962 and the optical sensor 5960.

FIG. 59E is a side cross-sectional view of a rotor 5910 including afirst fluid port 5912 (e.g., fluid conduit, inlet) and a second fluidport 5914 (e.g., fluid conduit, outlet). In some variations, the firstfluid port 5912 and the second fluid port 5914 may extend in parallelwith each other and/or a rotational axis of the rotor 5910. In somevariations, the first fluid port 5912 and the second fluid port 5914 maybe disposed on opposite sides of the rotor 5910, which may simplifyfluid routing, cartridge design, and also reduce manufacturing costs.For example, the fluidic seals may be simplified since they contain onlya single lumen each. Conventionally, a complicated fluid flow path(including inlet and outlet) is formed on a first side of a rotor due toa fixed mechanical coupling of a drive motor to a second side of therotor. FIGS. 59F and 59G are cross-sectional side views of a rotor 5910disposed within housing 5931.

FIG. 60A is a plan view of a rotor 6000 that may be used with any of theCCE systems, CCE modules, cartridges, housings, combinations thereof,and the like described herein. The rotor 6000 may comprise a first fluidconduit 6010, a cone 6020 (e.g., bicone), a second fluid conduit 6030, amagnetic portion 6040 (e.g., magnet), and housing 6050. Fluid may flowsequentially through the first fluid conduit 6010, the cone 6020, andthe second fluid conduit 6030. In some variations, the magnetic portion6040 may comprise one or more magnets. In some variations, the rotor6000 may define a rotation axis 6060. In some variations, at least aportion of the first fluid conduit 6010 and at least a portion of thesecond fluid conduit 6030 may extend parallel to the rotation axis(e.g., into and out of the page with respect to FIG. 60A). In somevariations, at least a portion of the first fluid conduit 6010 and atleast a portion of the second fluid conduit 6030 may be co-axial.

In some variations, the cone 6020 may comprise a bicone having a firstcone including a first base and a second cone including a second basesuch that the first base faces the second base. In some variations, abicone may comprise a cylinder (or some other shape) between and/or influid communication with the first cone and the second cone. Forexample, one or more cones of a rotor may comprise a generally steppedshape. For example, one or more cones may comprise stacked circularsteps. In some variations, a cone of a rotor may comprise a single cone.

In some variations, at least a portion of the rotor may be opticallytransparent to facilitate visualization and/or imaging of the rotor 6000and/or fluid (e.g., cells) in the rotor 6000. For example, the cone 6020may be transparent, as well as portions of the first fluid conduit 6010and the second fluid conduit 6030.

In some variations, the cone may comprise a volume of between about 10ml and about 40 ml. In some variations, the cone may comprise a coneangle of between about 40 degrees and about 60 degrees.

In some variations, a cone may comprise a first cone (e.g., distal cone)and a second cone (e.g., proximal cone) where the first cone is largerthan the second cone. In some variations, a first cone length may bebetween about 60 mm and about 90 mm. In some variations, a proximal conelength may be between about 15 mm and about 40 mm. In some variations, acone diameter (e.g., maximum diameter of the cone) may be between about15 mm and about 40 mm.

In some variations, the rotor 6000 may comprise an asymmetric shape. Insome variations, a first portion (e.g., first end) of the rotor 6000 maycomprise the cone 6020 and a second portion (e.g., second end) maycomprise a paddle shape.

In some variations, the cone may comprise a length of at least about 4cm (e.g., between about 9 cm and about 12 cm), a cone diameter of about5 cm or less (e.g., between about 3 cm and about 5 cm), a fluid flowrate of up to about 100 ml/min (e.g., between about 60 ml/min and about100 ml/min), and a rotation rate of less than about 3000 RPM. The shapeof the first cone and the second cone may be generally linear (asopposed to convex or concave).

FIGS. 60B and 60C are perspective views, and FIG. 60D is a side view ofa rotor 6002 comprising a first fluid conduit 6012, a cone 6022, asecond fluid conduit 6032, and a housing 6052. FIG. 60E is a perspectiveview of the rotor 6002 disposed in a housing 6090.

FIG. 60F is a plan view of a rotor 6004 having two cones (e.g., twobicones) configured to elutriate cells (e.g., red blood cells,leukapheresis product) in a second cone in order to recirculate a bufferfor reuse. The rotor 6004 may comprise a housing 6052, a first fluidconduit 6012, a first cone 6022 coupled to the first fluid conduit 6012,a second fluid conduit 6023 coupled to the first cone 6022, and a secondcone 6024 coupled to the second conduit 6023, and a third fluid conduit6032 coupled to the second cone 6024. The first cone 6022 may comprise afirst volume, and the second cone 6024 may comprise a second volumelarger than the first volume. In some variations, a ratio of a secondvolume to a first volume may be between about 2:1 to about 5:1. Fluidmay flow sequentially through the first fluid conduit 6012, the firstcone 6022, the second fluid conduit 6023, the second cone 6024, and thethird fluid conduit 6032. In some variations, the rotor 6004 maycomprise a magnetic portion 6042.

In some variations, the first cone 6022 may comprise a first bicone andthe second cone 6024 may comprise a second bicone. In some variations,the first bicone may comprise a third cone including a first base and afourth cone including a second base such that the first base faces thesecond base. In some variations, the second bicone may comprise a fifthcone including a third base and a sixth cone including a fourth basesuch that the third base faces the fourth base.

In some variations, a portion of the rotor 6004 may be opticallytransparent, such as first cone 6022, second cone 6024, and at least aportion of first fluid conduit 6012, second fluid conduit 6023, andthird fluid conduit 6032. In some variations, the first fluid conduit6012 may comprise an inlet and the third fluid conduit 6032 may comprisean outlet.

In some variations, cells may enter the first cone 6022 and red bloodcells (RBCs) 6030 may be elutriated into the second cone 6024. Since thesecond cone 6024 is further out from an axis of rotation (center ofhousing 6052), the RBCs 6030 may be concentrated at an inlet 6025 of thesecond cone 6024 due to centrifugation. The larger volume of the secondcone 6024 may further reduce the velocity of fluid (e.g., buffer),thereby reducing the force on RBCs 6030 within the second cone 6024. Byrecirculating the fluid (e.g., buffer), a higher concentration of RBCsmay be elutriated with less fluid (e.g., buffer). In some variations,white blood cells 6040 may be harvested from the first cone 6022. Anoptical sensor may be configured to image the first cone 6022 togenerate imaging data used to identify a boundary between the WBCs 6040and RBCs 6030. In some variations, the recirculating fluid may be passedthrough a filter to remove small particles (e.g., platelets) with lessfluid (e.g., buffer).

FIG. 60G is a plan view and FIG. 60H is a side view of a rotor 6005having two cones (e.g., two bicones) configured to elutriate cells(e.g., red blood cells) in a second cone. A rotor having two cones mayfacilitate recirculation of buffer for reuse. The rotor 6006 maycomprise a housing 6052, a first fluid conduit 6012, a first cone 6022coupled to the first fluid conduit 6012, a second cone 6024 coupled tothe first cone 6022, and a fluid conduit 6032 (e.g., outlet) coupled tothe second cone 6024.

FIG. 60I is a perspective view of a rotor 6006 comprising a cone 6024and housing 6054. FIG. 60J is a perspective view of a rotor 6007comprising a cone 6026 and housing 6056. FIG. 60K is a schematic planview of rotor 6008 and corresponding dimensions. FIG. 60L is an image ofa set of rotors having varying dimensions.

FIGS. 11A-11C depict another variation of the counterflow centrifugalelutriation (CCE) module 1100. FIG. 11A is a perspective view of acartridge 1110 comprising a CCE module 1100 in an extended configurationconfigured to receive a CCE instrument. FIGS. 11B and 11C arecross-sectional side views of a CCE module 1100 in respective retractedand extended configurations. In some variations, a CCE module maycomprise a conical element having an internal surface and an externalsurface fixedly attached to a distal end of a linear member having aninternal surface and an external surface. The proximal end of the linearmember may be rotationally attached to a fulcrum in order to enableextension, retraction, and/or rotation of the linear member. Forexample, FIG. 11C depicts a linear member extended outside the housingof the cartridge and then rotated to generate a centrifugal force. Acell product may be conveyed between the internal surface and externalsurface of the linear member (optionally in tubing) to the conicalelement and fed into an opening at the distal end of the internalsurface of the conical element, such that the flow of the cell productmay run counter to the centrifugal force generated by rotation of thelinear member. Cells in the cell product may be separated based on theratio of their hydrodynamic cross section to their mass, due to thecounterflow of the solution and sedimentation of cells subject tocentrifugal force. The flow rate may then be increased and/or therotation of the linear member may be decreased to permit cells toselectively return through the void in the interior surface of thelinear member to the proximal end of the linear member. The selectedcells may be directed into a tube that returns the selected cells to thecartridge. After an enrichment/washing step is performed, the linearmember may be retracted into the housing to the retracted configurationas shown in FIG. 11B.

Magnetic Cell Selection

Generally, the systems and methods described herein may select cells onthe basis of magnetically labeled cells corresponding to cells having apredetermined antigen. For example, a cell suspension of interest may beimmunologically labeled with magnetic particles (e.g., magnetic beads)configured to selectively bind to the surface of the cells of interest.The labeled cells may generate a large magnetic moment when the cellsuspension is flowed through a flow cell. The flow cell may be disposedin proximity to a magnet array (e.g., permanent magnets, electromagnet)generating a magnetic field having a gradient across the flow cell toattract the labeled cells for separation, capture, recovery, and/orpurification. The magnet array may be configured to generate non-uniformmagnetic fields at the edges and the interfaces of the individualmagnets so as to cover the full volume of the flow cell such that amagnetophoretic force equals a drag force exerted by the fluid flowingthrough the flow cell.

FIG. 61A-61C are schematic views of a magnetic cell separation (e.g.,magnetic-activated cell selection) system and process. A magnetic cellseparation system may comprise a flow cell 6110 comprising an inlet 6130and an outlet 6132, a magnet array 6120, a first fluid source 6140(e.g., input sample source), a second fluid source 6142 (e.g., buffersource), a third fluid source 6150 (e.g., target cell reservoir), afourth fluid source 6152 (e.g., waste reservoir), and a set of valves6134. As shown in step 6100, a set of cells 6160, 6170 may compriselabeled cells 6160 (e.g., magnetically labeled cells) and non-labeledcells 6170 may flow into the flow cell 6110. For example, a set of thecells 6160 may be labeled with a magnetic-activated cell selection(MACS) reagent. A MACS reagent may be incubated with the set of cells tolabel (e.g., attach, couple) the cells to the MACS reagent. As describedin more detail herein, the magnet array 6120 may be disposed external tothe flow cell 6110 such that the magnet array 6120 may be moveablerelative to the flow cell 6110. For example, the magnet array 6120 maymove away from the flow cell 6110 to facilitate flowing the set of cells6160 out of the flow cell 6110. Conventional flow cells comprisetortuous paths including meshes and/or beads to capture cells. However,recovery of labeled cells from conventional flow cell configurations isdifficult, By contrast, the flow cells 6110 described herein may lacktortuous paths such as beads, meshes, and the like, and therefore enableserial separations to be performed efficiently using either positiveselection or negative selection. In some variations, the flow cells maycomprise generally laminar channels as described in more detail herein.

At step 6102, the magnet array 6120 may magnetically attract the set ofcells 6160 towards the magnet array 6120 for a predetermined dwell timeand/or based on a measured quantity of magnetically separated cells. Insome variations, the dwell time may be at least one minute (e.g., atleast two minutes, at least three minutes, at least five minutes). Thenon-labeled cells 6170 are not magnetically attracted to the magnetarray 6120 and may flow out of the outlet 6132 of the flow cell 6110 andinto the fourth fluid source 6152. In some variations, the fluid (e.g.,cells 6160, 6170) within the flow cell may be held statically within theflow cell 6110 for a dwell time before the fluid (e.g., cells 6170) flowfrom outlet 6132. In some variations, a longitudinal axis of the flowcell 6110 may be oriented substantially perpendicular to ground in orderfor fluid flow through the flow cell 6110 to be aided by gravity. Atstep 6104, the magnetic coupling between the magnet array 6120 and thecells 6160 may be released after the dwell time, and the cells 6160 mayflow into the third reservoir 6150.

In some variations, stiction may cause cells to remain attached to asurface of a flow cell even after removal of a magnet array 6120.Therefore, a gas may be flowed through the flow cell 6110 to aid cellcollection into the third reservoir 6150. Gas flow through the flow cellmay provide improved cell recovery over liquid flushing through the flowcell. An interface generated by a gas (e.g., bubble, air gap) may bemaintained by gravity, thereby enabling implementation of a relativelywide flowcell that further improves cell recovery relative to ahorizontally oriented flow cell. The MACS modules described herein maybe configured for positive selection and/or negative selection bymodifying the sequence of steps.

Additionally or alternatively, an optical sensor may be configured toimage a flow cell to generate imaging data used to identify a quantityof cells magnetically attracted to the magnet array. Fluid containinglabeled cells may be flowed out of the flow cell when a predeterminedquantity of cells have been measured by the optical sensor.

FIG. 62A is a perspective view of a MACS module 6200 in a firstconfiguration. The MACS module 6200 (as well as any of the MACS modulesdescribed herein) may be a component of any of the cartridges describedherein. For example, a cartridge for cell processing may comprise aliquid transfer bus and a plurality of modules with each modulefluidically linked to the liquid transfer bus. The MACS module 6200 maycomprise a flow cell 6210 comprising an elongate cavity having a cavityheight, an inlet 6230, and an outlet 6232. The MACS module 6200 mayfurther comprise a magnet array 6220 comprising a plurality of magnets.Each of the magnets may be spaced apart by a spacing distance, such asillustrated in FIGS. 62G, 63D, and 63E, although FIGS. 62A-62Eillustrate a magnet array 6220 with magnets in contact with adjacentmagnets.

FIG. 62G is a schematic diagram of the flow cell 6210 and magnet array6220. In some variations, the flow cell 6210 may comprise a cavityheight 6202 and a cavity width 6204. Fluid may be configured to flowthrough the flow cell 6210 in a first direction 6206. The magnet array6220 may comprise a plurality of magnets with each magnet comprising arespective width 6222. In some variations, adjacent magnets may beseparated by a predetermined spacing distance 6224. Each magnet pair mayhave the same or different spacing distance 6224. As shown in FIG. 62G,an orientation (e.g., poles) of the magnets in the magnet array 6220 maycomprise a predetermined pattern.

In some variations, a ratio of the cavity height 6202 to the spacingdistance 6224 is between about 20:1 and about 1:20, between about 10:1and about 1:10, between about 5:1 and about 1:5, and between about 3:1and about 1:3, including all values and sub-ranges in-between. In somevariations, an actuator 6240 (e.g., linear, rotary) may be configured tomove the magnet array 6220 relative to the flow cell 6210. In somevariations, an orientation (e.g., poles) of the magnets in the magnetarray 6220 may comprise a predetermined pattern (e.g., Halbach array).

In some variations, the magnet array 6220 may move relative to the flowcell 6210 or vice versa. FIG. 62A illustrates the MACS module 6200 in anopen configuration and FIG. 62B illustrates the MACS module 6200 in aclosed configuration. FIG. 62B is a perspective view of the MACS system6200 in a second configuration where labeled cells may be magneticallyattracted towards the magnet array 6220. In the second configuration,the magnetic field lines generated by the magnet array traverse the flowchannel exerting a magnetophoretic force on magnetically tagged cellsthat are injected into the channel. FIG. 62C is a cross-sectional sideview of the MACS system 6200 including the magnet array 6220. FIG. 62Dis a perspective view of a MACS system 6200 in the second configuration.FIG. 62E is a plan view of a flow cell 6210 and magnet array 6220 of aMACS system. FIG. 62F is a plan view of a flow cell 6210 of a MACSsystem.

FIG. 63A-63E are perspective views of a set of magnet arrays 6300, 6310,6320, 6330, 6340. One or more of the size, strength, shape, spacing, andorientation of the magnets in a magnet array may be set to generate amagnetic field to attract magnetically-labeled cells. Additionally oralternatively, a magnet array may comprise a high-magnetic permeabilitymaterial configured to enhance or reduce the field strength and fieldgradients within the flow cell. The material may be disposed between amagnet and flowcell. Additionally or alternatively, the material may bedisposed within the flowcell and/or on one or more sides of theflowcell.

FIGS. 64A and 64B are respective perspective and cross-sectional sideviews of a MACS module 6400 comprising a flow cell 6410 and a magnetarray 6420. The flow cell 6410 may comprise a set of linear channels6412, 6414, 6416 comprising a first channel 6412 parallel to a secondchannel 6414, and a third channel 6416 in fluid communication with eachof the first channel 6412 and the second channel 6416. As shown in FIG.64B, the third channel 6416 may be disposed between the first channel6412 and the second channel 6416 and define a volume where fluid fromthe first channel 6412 and the second channel 6416 interact (e.g., mix).In some variations, the flow cell 6410 may comprise a first inlet 6430coupled to the first channel 6412 and configured to receive a firstfluid 6460 (e.g., cells). A second inlet 6431 may be coupled to thesecond channel 6414 and configured to receive a second fluid 6470 (e.g.,buffer). The flow cell 6410 may comprise a first outlet 6432 coupled tothe first channel 6412 and a second outlet 6433 coupled to the secondchannel 6414.

The magnet array 6420 may be disposed external to the flow cell 6400 andmay be moved relative to the flow cell 6400 as described herein. In somevariations, a longitudinal axis of the flow cell 6410 may beperpendicular to ground such that fluid flows in a generally verticaldirection.

In some variations, the first channel 6412 may have different dimensionsform the second channel 6414. For example, a first cavity height of thefirst channel 6412 may be larger than a second cavity height of thesecond channel 6414. For example, a ratio of the first cavity height toa second cavity height may be between about 1:1 to about 3:7, betweenabout 1:1 to about 2:3, and between about 2:3 to about 3:7, includingall values and sub-ranges in-between. Fluid flowing through the firstchannel 6412 may have a slower flow rate relative to the second channel6414 due to the larger cavity height of the first channel 6412 relativeto the second channel 6414. In some variations, the third channel 6416may comprise a ratio of a length of the third channel 6416 to a diameterof the third channel 6416 of between about 2:1 to about 6:1, betweenabout 2:1 to about 3:1, between about 3:1 to about 4:1, between about4:1 to about 5:1, between about 5:1 to about 6:1, and between about 3:1to about 5:1, including all values and sub-ranges in-between.

As shown in FIG. 64B, a first fluid 6462 may flow through the flow cell6410 generally following a first direction. The magnetically-labeledcells 6416 within the first fluid 6462 may separate from the rest of thefirst fluid 6462 within the third channel 6416 as the magneticattractive forces generated by magnet array 6420 pulls the cells 6416away from the first channel 6412 and towards the second channel 6414(e.g., towards the magnet array 6420). Similarly, a second fluid 6470(e.g., buffer) may flow through the second channel 6414. As the cells6416 flow towards the magnet array 6420, they displace the second fluid6470 flowing through the third channel 6416 such that a portion of thesecond fluid 6470 may flow into the first channel 6412. In this manner,magnetically-labeled cells 6416 may be magnetically separated from afirst fluid 6462 and the second fluid 6470 may aid removal of the firstfluid 642 not including the cells 6416.

In some variations, a set of fluidic loops may be coupled to the flowcell to enable a plurality of cell separation cycles. FIG. 64C is aschematic diagram of a MACS module comprising a flow cell 6410, a firstfluid conduit 6480 coupled to an inlet 6430 of the flow cell 6410 and anoutlet 6432 of the flow cell 6410. The first fluid conduit may 6480 maybe configured to receive the set of cells from an outlet 6432 of theflow cell 6410 for recovery and/or recirculation through the inlet 6430of the flow cell 6410. A second fluid conduit 6490 may be coupled to theinlet 6431 of the flow cell 6410 and the outlet 6433 of the flow cell6410 to recirculate fluid such as buffer and unrecoveredmagnetically-labeled cells. The second fluid conduit 6490 may beconfigured to receive a fluid without the set of cells from the flowcell 6410. Higher purities of labeled cells may be recovered based on anumber of cycles performed. For example, a single cell separation cyclemay yield about 80% cell purity, a second cell separation cycle mayyield about 96% cell purity, a third cell separation cycle may yieldabout 99.2% cell purity, and a fourth cell separation cycle may yieldabout 99.84% cell purity.

In some variations, applying a centrifugal force to a magnetic cellseparation process may further attract labeled cells toward a magneticarray independently of fluid flow rate so as to maintain throughput.FIGS. 65A-65C are schematic diagrams of a MACS module 6500 utilizingcentrifugal force to aid a cell separation process. FIG. 65A depicts aflattened flow cell 6510 configured to be wrapped to form a generallycylindrical shape 6512. The flow cell 6510 may comprise a curved flowpath 6520.

FIG. 65B illustrates a cylindrical flow cell 6510 concentricallysurrounded by (e.g., nested within) a cylindrical magnet array 6530. InFIG. 65B, only a cross-section of the magnet array 6530 is shown for thesake of clarity. The flow cell 6510 may be spaced apart from the magnetarray 6530 by a predetermined spacing distance. Accordingly, the flowcell 6510 may be configured to rotate 6550 about a longitudinal axis togenerate a centrifugal force on the fluid 6540 within the flow path 6520in an outward direction towards the magnet array 6530. During a cellseparation process, the fluid may be subject to set of forces depictedin FIG. 65C including a bulk fluidic force 6560 in an axial (e.g., bulkflow) direction, a centrifugal force 6570 in a radially outwarddirection from a center of rotation (e.g., proportional to a netparticle system buoyancy), and a magnetic force 6580 extending radiallyoutward from a center of rotation (e.g., proportional to net particlesystem magnetic attractiveness). In some variations, labeled cells maycomprise a higher density than non-labeled cells. Therefore, centrifugalforce may preferentially push the labeled cells towards the magnet 6530,further increasing the specificity and efficiency of cell separation.

FIGS. 66A-66C are schematic diagrams of a cell separation system andprocess. A magnetic cell separation system may comprise a flow cell 6610comprising a flow path 6620 (shown schematically flattened for sake ofclarity), and a magnet array 6630. As shown in step 6600, a set of cells6640, 6642 may comprise labeled cells 6640 (e.g., magnetically labeledcells) and non-labeled cells 6642 may flow into the flow path 6620 offlow cell 6610. For example, a set of the cells 6640 may be labeled witha magnetic-activated cell selection (MACS) reagent. The magnet array6630 may be disposed external to the flow cell 6610 such that the magnetarray 6630 may be moveable relative to the flow cell 6610. For example,the magnet array 6630 may move away from the flow cell 6610 tofacilitate flowing the set of cells 6640 out of the flow cell 6610.

At step 6602, the flow cell 6650 may be rotated to generate centrifugalforce to push the cells 6640, 6642 toward the magnet array 6630. In somevariations, a longitudinal axis of the flow cell 6610 may be orientedsubstantially perpendicular to ground in order for fluid flow throughthe flow cell 6610 to be aided by gravity. At step 6604, the magnetarray 6630 may magnetically attract the set of cells 6640 towards themagnet array 6630 for a predetermined dwell time as described herein.The non-labeled cells 6642 are not magnetically attracted to the magnetarray 6630 and may flow out of the flow cell 6610 into, for example, awaste vessel. In some variations, the fluid (e.g., cells 6160, 6170)within the flow cell may be held statically within the flow cell 6110for a dwell time before the fluid (e.g., cells 6170) flow from outlet6132. In some variations, the magnetic coupling between the magnet array6630 and the cells 6640 may be released after the dwell time, and thecells 6640 may be recovered.

FIGS. 12A and 12B illustrate the magnet of the MACS instrument 1200comprising a magnet and a MACS module 1210. The magnet is shown in FIG.12A in an ON configuration and shown in FIG. 12B in an OFFconfiguration.

Bioreactor

The bioreactors described herein may comprise a vessel configured toculture mammalian cells. Generally, cell and gene therapy products maybe grown in a bioreactor to produce a clinical dose which maysubsequently be administered to a patient. A number of biological andenvironmental factors may be controlled to optimize the proliferationspeed and success of cell growth. The bioreactor modules describedherein enable one or more of monitoring, adjusting, and/or controllingof cell growth (e.g., to facilitate consistent and efficient cellularproliferation).

FIG. 67A is a schematic diagram of a cell processing system 6700 (e.g.,bioreactor module) comprising one or more of a bioreactor 6710, one ormore sensors 6720, an agitator 6730, a temperature regulator 6740, and agas regulator 6750. In some variations, the sensor 6720 may beconfigured to monitor (e.g., measure, sense, determine) one or morecharacteristics of the bioreactor module 6700 and cells in thebioreactor 6710. For example, the sensor 6720 may comprise one or moreof a pH sensor, a dissolved oxygen (DO) sensor, a temperature sensor, aglucose sensor, a lactose sensor, a cell density sensor, a humiditysensor, combinations thereof, and the like. One or more of the sensorsmay be a non-invasive optical sensor.

FIGS. 67B-67D are schematic diagrams of a cell processing systemcomprising a workcell 6760, a bioreactor system 6700 (e.g., bioreactorinstrument), a cartridge 6770, an agitator 6730, and a fluid connector6780. In some variations, a cartridge 6770 for cell processing maycomprise a liquid transfer bus and a plurality of modules (e.g.,bioreactor module, CCE module, MACS module, EP module). Each module maybe fluidically linked to the liquid transfer bus. The bioreactor modulemay comprise at least one bioreactor.

The bioreactor instrument 6700 may be configured to interface with thecartridge 6770. In some variations, the bioreactor instrument 6700 maycomprise the agitator 6730 configured to couple to the bioreactor. Theagitator may be configured to agitate cell culture media comprisingcells. In some variations, the fluid connector 6780 may be configured tocouple the bioreactor system 6700 and workcell 6760.

FIG. 67B depicts a cartridge 6770 comprising a bioreactor disposedwithin a workcell 6760. The bioreactor 6700 may be uncoupled from theworkcell 6760. Once the fluid connector 6780 couples (e.g., to create asterile flow path) the workcell 6760 to the bioreactor 6700, thecartridge 6770 may be moved into the bioreactor 6700, as shown in FIG.67C. For example, the cartridge 6770 may be coupled to (e.g., disposedon) an agitator 6730 and then agitated, as shown in FIG. 67D. In somevariations, the fluid connector 6780 may comprise a set of foldablesidewalls (e.g., like an accordion) configured to receive and dissipatethe agitation of the agitator 6730 without transmitting such motion tothe workcell 6760. That is, the fluid connector 6780 may function as abellows to maintain the connection between the workcell 6760 andbioreactor 6700 without agitating the workcell 6760. In some variations,the fluid connector 6780 may couple the bioreactor (e.g., of cartridge6770) to a liquid transfer bus.

In some variations, an agitator may be configured to generate motion(e.g., orbital, rotary, linear) to the bioreactor in order to mix theculture in instances where it is required to encourage interactions witha reagent and cells. For example, orbital motion may be used to create ahomogenous culture volume such that a small sample taken from theculture may be representative of the culture at large. In somevariations, the agitator 6730 may comprise one or more impellers. Theagitator 6730 may be configured to provide variable-intensity mixingduring culture at defined periods.

In some variations, orbital motion may encourage increased interactionswithin the cell culture, such as in the toroidal bioreactors describedherein that comprise a geometry that may encourage the continuous andgentle flow of fluid around the bioreactor, thereby aiding homogenousmixing with minimal shear stress transferred to the cells.

In some variations, the temperature regulator 6740 may be configured tocontrol a temperature of a bioreactor and corresponding processes. Thetemperature regulator 6740 may be coupled to the bioreactor. Forexample, the temperature regulator 6740 may control a temperature of acell culture to be between about 2° C. and about 40° C. and therebyensure that a culture is heated to physiological conditions and cooledto slow metabolic processes (e.g., to keep cells in a dormant state) asdesired. For example, the thermal regulator 6740 may comprise acirculating coolant coupled to a heat exchanger coupled to a thermalinterface (e.g., heating/cooling plate).

In some variations, the gas regulator 6750 may be coupled to thebioreactor and configured to control a gas composition of a bioreactorand corresponding processes using one or more of Clean Dry Air (CDA),carbon dioxide, and nitrogen. The gas regulator 6750 may be coupled tothe bioreactor. For example, the sensors 6720 and gas regulator 6750 mayprovide closed-loop gas control of the bioreactor module 6700. In somevariations, CDA may comprise oxygen such as pure oxygen. In somevariations, the gas regulator may comprise a manifold coupled to one ormore gas sources. The manifold may include a solenoid coupled to a valve(e.g., restrictive orifice) configured to control gas flow through thebioreactor 6710. The solenoid may be configured to pulse to control aquantity and composition of gas received through the manifold.Additionally or alternatively, one or more of a proportional valve andMass Flow Controller (MFC) may be configured to meter and control theflow of gas to a manifold. In some variations, the gas regulator 6750may comprise one or more sensors to measure the gas mixture and/or flowrate. Additionally or alternatively, the sensors may be configured forclosed-loop control of gas flow through the gas regulator.

In some variations, measured pH from a pH sensor may be used to controla pH of the bioreactor 6710 using the gas regulator 6750. For example,in response to the measured pH, gas regulator 6750 may control a CO₂concentration of the gas contacting the cell culture to control the freehydrogen ions and pH of the culture. In some variations, a pH of thebioreactor 6710 may be between about 5.5 and about 8.5. One or more ofCO₂ composition of the gas in the bioreactor 6710, buffer, and reagents(e.g., acid, base) may be used to regulate pH. In some variations, adissolved oxygen concentration of the bioreactor 6710 may be betweenabout 0% and about 21%. Nitrogen composition of the gas in thebioreactor 6710 may be used to regulate the dissolved oxygenconcentration. For example, control of both the agitator in thebioreactor and the flow rate and composition of the gas contacting thecell culture may regulate the dissolved carbon dioxide concentration.

In some variations, measured dissolved oxygen from a dissolved oxygensensor may be used to control an oxygen concentration (e.g., belowatmospheric levels) of the bioreactor 6710 using the gas regulator 6750.For example, gas regulator 6750 may control a nitrogen concentration ofthe gas contacting the cell culture to create hypoxic conditions.

FIGS. 68A and 68B are cross-sectional perspective views of a bioreactor6800 comprising an enclosure 6810 comprising a base 6812, a sidewall6814, and a top 6816. A gas-permeable membrane 6820 may be coupled toone or more of the base 6812 and the sidewall 6814 of the enclosure6810. In some variations, the enclosure 6810 may comprise a firstchamber 6830 having a first volume and a second chamber 6832 having asecond volume, the first chamber 6830 separated from the second chamber6832, and the first volume smaller than the second volume. In somevariations, the first chamber 6830 may be concentrically nested withinthe second chamber 6832. For example, nesting the chambers may enablelarger overall working volume ranges (e.g., 100:1). The first chamber6830 may comprise a well shape with an angled base surface to promotefluid pooling at a center of the first chamber 6830 during aspiration.In some variations, the base 6812 may be disposed on a thermal regulator(not shown) such as a thermoelectric element. In some variations, theenclosure 6810 may be composed of a thermally conductive material suchas a metal (e.g., aluminum).

In some variations, the bioreactor 6800 may be coupled to a gasregulator (not shown) to facilitate gas transfer through thegas-permeable membrane 6820 (e.g., into and out of the culture). Thegas-permeable membrane 6820 may be configured to hold a cell culture.Gas may diffuse through the surfaces of the culture that contact thegas-permeable membrane to enable increased oxygenation of the cellculture and removal of gaseous metabolic byproducts of the cell culture,and thus increase the potential for metabolic activity. For example, thegas-permeable membrane 6820 enables dissolved oxygen to diffuse into theculture in close proximity to a cell bed where the oxygen may beconsumed. In some variations, the bioreactor may be coupled to both afirst gas regulator to facilitate gas transfer through the gas-permeablemembrane and a second gas regulator to facilitate control of headspacegas composition.

In addition to gas transfer, the bioreactors described herein may beconfigured to efficiently control a temperature of a cell culture usinga conductive thermal interface (e.g., gas-permeable membrane 6820,enclosure 6810) along both a base and sidewall of the bioreactor.

In some variations, the first chamber 6830 may comprise a working volumeof between about 10 ml and about 100 ml. In some variations, the firstchamber 6830 may comprise a total volume of between about 10 ml andabout 130 ml. In some variations, the second chamber 6832 may comprise aworking volume of between about 100 ml and about 1000 ml. In somevariations, the second chamber 6832 may comprise a total volume ofbetween about 100 ml and about 1400 ml. In some variations, the firstchamber 6830 may comprise a diameter of between about 10 mm and about100 mm, and a height of between about 10 mm and about 100 mm. In somevariations, the second chamber 6832 may comprise a diameter of betweenabout 100 mm and about 250 mm, and a height of between about 10 mm andabout 100 mm.

As shown in FIG. 68B, a base 6822 of the gas-permeable membrane 6820 maycomprise an angle between about 3 degrees and about 10 degrees relativeto the base 6812 of the enclosure 6810. Similarly, FIGS. 69A and 69Bdepict a sloped base. For example, due to a slope of the base 6822, thechambers 6830, 6832 are deeper towards a center of the bioreactor 6800.This may encourage cell growth towards a center of the bioreactor 6800,which may aid one or more of cell sampling, cell transfer, cellrecovery, and the like. In some variations, orbital motion of thebioreactor 6800 may promote cell congregation toward a center of thebioreactor 6800, thereby increasing interaction between the cells.

In some variations, the gas-permeable membrane 680 may comprise a curvedsurface. In some variations, the gas-permeable membrane may comprise aset of patterned curved surfaces. For example, the set of patternedcurved surfaces may comprise a radius of curvature of between about 50mm and about 500 mm.

In some variations, the bioreactor may be configured to facilitatemonitoring (e.g., temperature, pH, dissolved oxygen) and fluid flow(e.g., gas composition, fluid transfer) between the chambers. As shownin FIG. 68C, the enclosure 6810 may comprise one or more nested surfacescurved around a longitudinal axis (e.g., center) of the enclosure 6810.For example, the nested surfaces may comprise a set of concentrictoroids. The enclosure 6810 may comprise a toroid shape. FIG. 68C is aperspective view and FIG. 68D is a bottom view of enclosure 6810comprising a set of apertures 6818 (e.g., holes, openings, slits,slots). In some variations, the apertures 6818 may enable gas and/orheat transfer between the components and chambers of the bioreactor6800. Additionally or alternatively, one or more sensors may be coupledto the apertures 6818. For example, the apertures 6818 may be coupled toa non-contact sensor (e.g., pH, DO) such as an optical sensor (notshown) configured to determine a fluorescent spot disposed on a surfaceof the bioreactor. In some variations, one or more of a sensor and fluidconnector may be introduced through the apertures 6818.

In some variations, the gas-permeable membrane extends along the base6812 and the sidewall 6814 of the enclosure 6810, as shown in FIG. 68B.In some variations, the gas-permeable membrane extends only along thebase 6812 of the enclosure 6810. FIG. 68E is a perspective view and FIG.68F is a side view of the gas-permeable membrane 6820 where an outersurface of the gas-permeable membrane 6820 comprises one or moreprojections 6824 (e.g., projections, spacers, ribs). The projections6824 are also depicted in the perspective view of FIG. 68G and bottomview of FIG. 68H. The projections 6824 contact the enclosure 6810 anddefine a cavity between the enclosure 6810 and the gas-permeablemembrane 6820. That is, the projections 6824 may be configured tomechanically space away the enclosure 6810 from a portion of thegas-permeable membrane 6820 to facilitate thermal transfer from theenclosure 6810 to the cell culture. In some variations the gas-permeablemembrane may comprise polydimethylsiloxane (PDMS) (e.g., silicone),fluorinated ethylene propylene (FEP), polyolefin (PO), polystyrene (PS),ethyl vinyl acetate (EVA) and have a thickness of between about 0.1 mmand about 0.4 mm, between about 0.2 mm and about 0.3 mm, and about 0.25mm, including all ranges and sub-values in-between.

FIG. 69A is a cross-sectional side view of an enclosure 6910 of abioreactor comprising a first chamber 6912, a second chamber 6914, and acolumn 6916 extending along a longitudinal axis of the enclosure 6910.FIG. 69B is a cross-sectional perspective view of the enclosure 6910showing the nested curves of the enclosure 6910. The column 6916 may beconfigured to promote cell culture in combination with agitation such asorbital motion.

FIG. 70 is an exploded perspective view of a bioreactor 7000 comprisingan enclosure 7010, a gas-permeable membrane 7020, and a top 7030. Thetop 7030 may be composed of a material such as polyethylene.

FIG. 71A is a plan view of a bioreactor 7100 comprising a first chamber7110 and a second chamber 7120. FIG. 71B is a cross-sectional side viewof the bioreactor 7100.

FIGS. 13A and 13B are perspective views of a cartridge 1300 andbioreactor instrument interface 1310. The bioreactor instrumentinterface 1310 is coupled to the cartridge 1300 in FIG. 13B.

FIG. 14 is a perspective view of a bioreactor instrument 1410 comprisinga set of cartridges 1400, 1402, 1404 and cavities 1420, 1422, 1424configured to receive a respective cartridge. In some variations, eachcartridge may be docked to enable simultaneous expansion, culturing, orresting steps.

Electroporation Module

In some variations, an electroporation module may be configured tofacilitate intracellular delivery of macromolecules (i.e., transfectionby electroporation). An electroporation module may contain a continuousflow or batch mode chamber and one or more sets of electrodes forapplying direct or alternating current to the chamber. An electricaldischarge from one or more capacitors, or current sources, may generatesufficient current in the chamber to promote transfer of apolynucleotide, protein, nucleoprotein complex, or other macromoleculeinto the cells in the cell product. As with other modules describedherein, one or more components used for the process step (here,electroporation) may be provided on the cartridge or in the instrumentto which the cartridge interfaces. For example, the capacitor(s) and/orbatteries may be provided in the module on the cartridge or in theinstrument. The electroporation module may, in some variations, beconfigured to apply an electric field to a cell suspension undercontinuous flow in a microfluidic device, e.g., as described in Garciaet al. Sci. Rep. 6:21238 (2016).

Additionally or alternatively, intracellular delivery of macromoleculesmay also be achieved by other methods, such as mechanoporation. Itshould be understood that throughout the disclosure variationscomprising an electroporation module may instead or in addition comprisea mechanoporation module, or another module configured to perform anysuitable method of delivering macromolecules into cells. Mechanoporationcan be achieved by, for example, applying transient, fluidic pressure toa solution containing cells, or by applying physical pressure to thecells (e.g., by microneedles). Illustrative methods of mechanoporationby passing a cell suspension through a constriction are provided, e.g.,in International Patent Publication No. WO 2017/041051 and WO2017/123663, and are incorporated by reference herein. Mechanoporationcan also be achieved by applying a vortex to a cell suspension in amicrofluidic device.

FIG. 72 is a schematic diagram of an electroporation module 7200 (e.g.,electroporation system) comprising an electroporation chamber 7210(which may comprise a fluid conduit), a pump 7220, an inlet 7230, anoutlet 7232, a set of pinch valves 7234, a first fluid source 7240(e.g., fluid reservoir, cell reservoir), a second fluid source 7242(e.g., vent, gas source), a set of sensors 7250 (e.g., bubble sensors),and a controller (e.g., processor and memory) configured to control themodule 7200, and a signal generator 7270 configured to deliver anelectroporation signal (e.g., voltage pulse) to the electroporationchamber 7210.

In some variations, the fluid conduit 7210 may be configured to receivea first fluid comprising cells and a second fluid. A set of electrodesmay be coupled to the fluid conduit 7210. A pump may be coupled to thefluid conduit 7210. The controller 7260 may be configured to generate afirst signal to introduce the first fluid into the fluid conduit 7210using the pump 7220, generate a second signal to introduce the secondfluid into the fluid conduit 7210 such that the second fluid separatesthe first fluid from a third fluid, and generate an electroporationsignal to electroporate the cells in the fluid conduit 7210 using theset of electrodes.

In some variations, the second fluid may comprise a gas or oil. In somevariations, the controller may be configured to generate a third signalto introduce the third fluid into the fluid conduit 7210. The thirdfluid may be separated from the first fluid by the second fluid. In somevariations, a cartridge for cell processing may comprise a liquidtransfer bus and a plurality of modules such as the electroporationmodule 7200. Each module may be fluidically linked to the liquidtransfer bus.

The set of sensors 7250 may be configured to measure fluid changes in afluid conduit such as a change from a first fluid to a second fluid(e.g., liquid to air) in the fluid conduit. The module 7200 may furthercomprise a set of valves configured to ensure fluid does not backflowinto the electroporation chamber 7210 and/or fluid source 7240. Theelectroporation chamber 7210 may comprise a cavity configured to hold afluid to be electroporated and a set of electrodes to apply anelectroporation signal to the fluid. For example, the signal generator7270 may generate a square valve pulse as described in more detailherein.

In some variations, the electroporation module 7200 (e.g., valves 7234,pump 7220, sensors 7250, and controller 7260) may be configured tocontrol fluid flow through the electroporation chamber 7210 in adiscontinuous (e.g., batch process) manner. For example, a first batchof cells may undergo electroporation and be physically separated from asecond batch of cells by an intermediate fluid such as air or fluid suchas oil. Separating cell batches may reduce mixing of transfected andnon-transfected cells, and further ensure fixed batch volume. That is, afluid gap may form a visually verifiable boundary between cell batchesto reduce diffusion and mixing between electroporated andnon-electroporated cells. Separating cell batches may reduce theduration of time that cells are exposed to certain cytotoxic reagents(e.g., electroporation buffer), thereby increasing performance.

In some variations, a batch of cells may be electroporated whensubstantially static (e.g., substantially no fluid flow state). Bycontrast, conventional continuous flow electroporation has an upperfluid flow rate limit correlated to a transfection efficiency. In thebatch processing described herein, cell batches may be transferred intoand out of the electroporation chamber 7210 at a predetermined rate toincrease the overall throughput of the system 7200 without a decrease inelectroporation efficiency. Furthermore, the electroporation system 7200does not utilize a precisely controlled flow rate/pulse rate such asthose needed for continuous flow electroporation systems.

FIG. 73 is an exploded perspective view of an electroporation module7300 may comprise ah electrode 7310, a fluid conduit 7320 (e.g.,electroporation chamber), a substrate 7330 (e.g. alloy busbar), ahousing 7340, and a fastener 7350. In some variations, the fluid conduit7320 may be configured to hold a volume of fluid between about 0.4 mland about 3.5 ml. The electroporation module 7300 is a parallel-platedesign. In some variations, the electrodes may comprise stainless steeland may be separated by an insulating gasket. In some variations, theelectrodes may be polished and/or coated with nonreactive materials(e.g., gold, platinum) to reduce gradual buildup of biological matter(e.g., charged molecules, DNA, proteins) on the electrode surface.

Generally, a method of electroporating cells may comprise receiving afirst fluid comprising cells in a fluid conduit, receiving a secondfluid in the fluid conduit to separate the first fluid from a thirdfluid, applying an electroporation signal to the first fluid toelectroporate the cells. In some variations, the third fluid may bereceived in the fluid conduit separated from the first fluid by thesecond fluid. In some variations, the first fluid may be substantiallystatic when applying the electroporation signal.

FIGS. 74A-74B are schematic diagrams of variations of an electroporationprocess 7400, 7402. A method 7400 may include loading cells 7410 into anelectroporation chamber 7450. For example, at step 7412, a first fluidmay be pumped into the electroporation by opening valve v1 and the pumpgenerating negative pressure (valves v2 and v3 are closed). At step7414, a second fluid (e.g., gas, oil) may separate the first fluid froma third fluid to create a first batch of cells to electroporate. Forexample, valves v1 and v3 may be closed with valve v2 open and the pumpgenerating negative pressure. In some variations, a loading volume maybe between about 1 ml and about 3 ml with a pumping time of betweenabout 8 seconds and about 15 seconds (at a rate of about 20 ml/min). Atstep 7420, the cells of the first fluid may be electroporated with eachof the valves closed and the pump off. At step 7430, the cells of thefirst fluid may be flowed out of the electroporation chamber 7450 tooutput where valves v1 and v2 are closed, valve v3 is open, and the pumpgenerates positive pressure.

FIG. 74B depicts another configuration where a pump is disposed betweenan input and the electroporation chamber such that the pump may beconfigured to pump in a single direction. A method 7402 may includeloading cells 7411 into an electroporation chamber 7450. For example, atstep 7416, a first fluid may be pumped into the electroporation byopening valve v1 and v4, and the pump generating positive pressure(valves v2 and v3 are closed). At step 7418, a second fluid (e.g., gas,oil) may separate the first fluid from a third fluid to create a firstbatch of cells to electroporate. For example, valves v1 and v3 may beclosed with valves v2 and v4 open, and the pump generating positivepressure. At step 7422, the cells of the first fluid may beelectroporated with each of the valves closed and the pump off. At step7432, the cells of the first fluid may be flowed out of theelectroporation chamber 7450 to output where valves v1 and v4 areclosed, valves v2 and v3 are open, and the pump generates positivepressure.

In some variations, an impedance/resistance across electrodes of anelectroporation system may increase over time due to electrodepassivation/degradation due to charged biological matter (e.g., chargedmolecules, DNA, proteins) attaching to the electrode surface. Activeelectrical field compensation may be applied to ensure a consistentelectrical field strength applied to cells over multiple batches ofcells. This may reduce the need for electrode surface modification toreduce passivation.

FIG. 75 is a circuit diagram of a resistor divider network for anelectroporation process 7500. For example, a set of cells may beintroduced into an electroporation chamber 7510 to which a voltageV_(chip)) may be applied. Fluid resistance R_(b) corresponds to a fluid(e.g., cell mixture) resistance. Assuming a uniform cell distribution,the fluid resistance R_(b) should be consistent, also assuming the samevolume of each fluid batch being electroporated. R_(i) corresponds to aresistance between fluid and electrode, which increases over timethrough the electroporation process. In a conventional electroporationprocess, voltage V_(ps) is constant. However, due to the increasingR_(i) over time, the voltage applied to the fluid will decrease overtime, leading to lower electrical field strength.

Due to variations in fluid resistance R_(b) and the low number of pulsesthat may be applied, interpolation to compensate for reduced electricalfield strength may not accurately compensate for electrode passivation.

In some variations, a method of electroporating cells may comprisereceiving a first fluid comprising cells in a fluid conduit, applying aresistance measurement signal to the first fluid using a set ofelectrodes, measuring a resistance between the first fluid and the setof electrodes, and applying an electroporation signal to the first fluidbased on the measured resistance. In some variations, a second fluidcomprising a gas may be received in the fluid conduit before applyingthe electroporation signal to the fluid. The first fluid may beseparated from a third fluid by the second fluid.

FIGS. 76A-76D are plots 7600, 7602, 7604, 7606 of measurement waveformsand electroporation waveforms. FIG. 76A depicts a first resistancemeasurement pulse 7620 with a low voltage and a wide pulse width. FIG.76B depicts a second resistance measurement pulse 7622 with a highvoltage and a short pulse width. FIG. 76C depicts a third resistancemeasurement pulse 7624 with a continuous low voltage waveform to monitoran impedance change continuously over time. FIG. 76D depicts a fourthresistance measurement pulse 7626 with a low AC voltage waveform tomonitor an impedance change continuously over time. Each of theresistance measurement pulses avoid inducing electroporation in thecells by reducing voltage and/or pulse width. By monitoring the voltagecurrent of the applied resistance measurement pulse, a change inresistance may be measured and the electroporation pulse applied to acell batch may be compensated accordingly.

In some variations, an electroporation signal may comprise between about1 pulse and about 50 pulses, a voltage of between about 100 V and about700 V, a pulse width of between about 100 μs and about 1 ms, a pulsespacing between about 5 second to about 30 seconds, a resistance pulsevoltage of between about 10 V and about 40 V, and a resistance pulsewidth of between about 10 μs and about 50 μs.

For example, an eight-batch electroporation run may receive oneelectroporation pulse per batch. Each electroporation pulse may have anelectrical field strength between about 0.5 kV/cm and about 2.0 kV/cm.The resistance measurement pulse applied before each batch may have anelectrical field strength less than about 0.2 kV/cm such thatelectroporation is not induced by the resistance measurement pulse.

Sterile Liquid Transfer Device

Generally, the sterile liquid transfer devices described herein may beconfigured to store fluid for transfer to another component of a cellprocessing system such as a cartridge, bioreactor, and the like. In somevariations, the sterile liquid transfer device may comprise a portableconsumable configured to be moved using a robot. For example, a robotmay be configured to move a sterile liquid transfer device from areagent vault to an ISO 7 space to a sterile liquid transfer instrumentwithin a cell processing system. The sterile liquid transfer deviceenables the transfer of fluids in an automated, sterile, and meteredmanner for automating cell therapy manufacturing.

FIGS. 103A and 103B are perspective views of a sterile liquid transferdevice 10300 comprising a fluid cavity 10310 (e.g., container, vessel),fluid connector 10320 (e.g., fluid connector), and pump 10330. Fluidstored within fluid cavity 10310 may be transferred in and out of thesterile liquid transfer device 10300 through the fluid connector 10320using the pump 10330. In some variations, the sterile liquid transferdevice 10300 may comprise an engagement feature 10340 (e.g., robotmount) to facilitate robotic arm control.

Fluid Connector

Generally, the aseptic fluid connectors described herein may form asterile fluid pathway between at least two fluid devices to enable fluidtransfer that may be one or more of sterile, fully automated, andprecisely metered (e.g., precise control of a transferred fluid volume).In some variations, the robot may be configured to couple a fluidconnector between at least two of the plurality of instruments and oneor more cartridge. In some variations, the robot may be configured tooperate the fluid controller to open and close a set of ports and valvesof the fluid connector. The use of a robot and controller to operate thefluid connector may facilitate automation and sterility of a cellprocessing system.

In some variations, a system may comprise a robot configured to operatea fluid connector as described herein, and a controller comprising amemory and processor. The controller may be coupled to the robot. Thecontroller may be configured to generate a port signal to couple thefirst port to the second port using the robotic arm, generate a firstvalve signal to translate the first valve relative to the second valveusing the robotic arm, and generate a second valve signal to transitionthe first valve and the second valve to the open configuration.

In some variations, a fluid pump may be coupled to the sterilant source,and the controller may be configured to generate a first fluid signal tocirculate a fluid into the chamber through the sterilant port. Thecontroller may be configured to generate a second fluid signal tocirculate the sterilant into the chamber through the sterilant port tosterilize at least the chamber. The controller may be configured togenerate a third fluid signal to remove the sterilant from the chamber.

In some variations, the controller may be configured to generate a portsignal to couple the first port to the second port using the roboticarm, generate a first valve signal to translate the first valve relativeto the second valve using the robotic arm, and generate a second valvesignal to transition the first valve and the second valve to the openconfiguration.

The fluid connector may further allow for a plurality of connectioncycles in a sterile system and may be controlled without humanintervention. For example, the fluid connector may comprise one or moreof engagement features to facilitate robotic arm control and alignmentfeatures to ensure proper connection between connector components. FIG.15 is a block diagram of an illustrative variation of a fluid connectorsystem 1500 comprising a fluid connector 1510, first fluid device 1520,second fluid device 1522, sterilant source 1530, fluid source 1532,robot (e.g., robotic arm) 1540, and controller 1550. The fluid connector1510 may be removably coupled (e.g., connected/disconnected,attached/detached) to each of the first fluid device 1520, second fluiddevice 1522, sterilant source 1532, fluid source 1532, and robot 1540.In some variations, a fluid device may comprise one or more of acartridge and sterile liquid transfer device. For example, a sterileliquid transfer device may be in fluid communication with a cartridgevia the fluid connector. As described in more detail herein, separateportions (e.g., male connector, female connector) of the fluid connector1510 may be removably coupled to each other. The robot 1540 may beconfigured to physically manipulate (e.g., removably couple) one or moreof the fluid connector 1510, first fluid device 1520, second fluiddevice 1522, sterilant source 1530, and fluid source 1532 in apredetermined manner. For example, the robot 1540 may connect the fluidconnector 1510 between the first fluid device 1520 and the second fluiddevice 1522. The robot 1540 may also connect the sterilant source 1530and/or fluid source 1532 to a sterilant port of the fluid connector1510. In some variations, the robot 1540 may control one or more valvesand/or ports of the fluid connector 1510, and thereby initiate asterilization process for one or more portions of the fluid connector1510 using, for example, sterilant from the sterilant source 1530. Thecontroller 1550 may be coupled to one or more of the robot 1540,sterilant source 1530, and fluid source 1532 to control one or more offluid transfer and sterilization.

FIG. 16A is a schematic diagram of an illustrative variation of a fluidconnector 1600. The fluid connector 1600 may comprise a lumen extendingalong its length and be disposed between a first fluid device 1630 and asecond fluid device 1640 to enable fluid flow through the fluidconnector 1600. In some variations, the first fluid device 1630 andsecond fluid device 1640 may be aseptically connected and disconnectedusing the fluid connector 1600. The fluid devices 1630, 1640 maycomprise a closed sterile device, and may be the same or different typesof fluid devices. For example, the fluid devices 1630, 1640 may compriseone or more of a sterile liquid transfer device and consumable. In somevariations, the fluid connector 1600 may comprise a first connector 1610including a first proximal end 1612 and a first distal end 1614. Thefirst proximal end 1612 may be configured to couple to the first fluiddevice 1630. The first distal end 1614 may include a first port 1616,first housing 1617, and a first valve 1618. The first housing 1617 maybe configured to receive the first port 1616 in a closed configurationas described in more detail herein.

The fluid connector 1600 may further comprise a second connector 1620including a second proximal end 1622 and a second distal end 1624. Thesecond proximal end 1622 may be configured to couple to the second fluiddevice 1640. The second distal end 1624 may include a second port 1626,second housing 1627, and a second valve 1628. The second housing 1627may be configured to receive the second port 1626 in a closedconfiguration. In FIG. 16A, the first connector 1610 comprises asterilant port 1650 configured to couple to a sterilant source (notshown). Additionally or alternatively, the second connector 1620 maycomprise the sterilant port 1650. The sterilant port 1650 may beconfigured to be in fluid communication with the first distal end 1614and the second distal end 1624 when the second port 1626 is coupled tothe first port 1616 as described in more detail herein.

In some variations, a fluid device 1630, 1640 may comprise a sterilantchamber and a sterilant port configured to receive a sterilant. Thesterilant chamber may enclose a fluid device connector (not shown)configured to couple to a proximal end of a first connector 1610 orsecond connector 1620. The fluid device 1630, 1640 may receive asterilant in a similar manner as the fluid connector 1600.

FIG. 16B is a detailed schematic diagram of the first connector 1610including a first port housing 1617 and a chamber 1615. The chamber 1615may be defined by the cavity enclosed by one or more of the distal ends1614, 1624. For example, the chamber 1615 in FIG. 16B may comprise theportion of the first connector 1610 between the first valve 1618 and thefirst port 1616 in the closed configuration (e.g., the first distal end1614). In some variations, the first chamber 1615 may comprise a volumeof between about 1 cm³ and about 5 cm³. When the first connector 1610 iscoupled to the second connector 1620 and the ports 1616, 1626 are in anopen configuration (as shown in FIG. 16D), the chamber 1616 may comprisethe portion of the fluid connector 1600 between the first valve 1618 andthe second valve 1628 (e.g., the first distal end 1614 and second distalend 1624). The chamber 1615 may comprise an enclosed volume configuredto receive a fluid such as a sterilant from the sterilant port 1650. Insome variations, the sterilant port 1650 may comprise an inlet 1652 andoutlet 1654. Methods of using a fluid connector are described in moredetail with respect to FIGS. 16C-16L and 27.

In some variations, the fluid connector 1600 may comprise one or morealignment features and robot engagement features configured tofacilitate robotic manipulation, as described in more detail herein. Insome variations, the fluid connector 1600 may be coupled to one or moresensors, pumps, and valves to facilitate fluid transfer and monitoring.

In some variations, the components of the fluid connector in contactwith fluid may be USP Class VI compatible for cell processing and/or GMPapplications. In some variations, the components of the fluid connectormay be composed of a material including, but not limited to, one or moreof cyclic olefin copolymer (COC), polychlorotrifluoroethylene,polyetherimide, polysulfone, polystyrene, polycarbonate, polypropylene,silicone, polyetheretherketone, polymethylmethacrylate, nylon, acrylic,polyvinylchloride, vinyl, phenolic resin, petroleum-derived polymers,glass, polyethylene, terephthalate, metal, stainless steel, titanium,aluminum, cobalt-chromium, chrome, silicates, glass, alloys, ceramics,carbohydrate polymer, mineraloid matter, and combinations or compositesthereof.

FIGS. 17A-18D depict external and internal views of variations of afluid connector. FIG. 17A is a front perspective view of a fluidconnector 1700 in a closed port configuration. FIG. 17B is a rearperspective view and FIG. 17C is a rear view of the fluid connector1700. Generally, the fluid connector may comprise a plurality ofinternal seals to reduce contamination and aid sterilization, as well asalignment features to aid proper registration of the fluid connectorcomponents.

The fluid connector 1700 may comprise a lumen extending along itslength. In some variations, the fluid connector 1700 may comprise afirst connector 1710 including a first proximal end 1712 and a firstdistal end 1714. The first proximal end 1712 may be configured to coupleto a first fluid device (not shown for the sake of clarity). The firstproximal end 1712 may comprise a Luer connector or any other suitableconnector. The first distal end 1714 may include a first port 1716 andfirst housing 1717. The first housing 1717 is shown in FIG. 17A holdingthe first port 1716 in a closed configuration. The first connector 1710further comprises a sterilant port 1750, 1752 configured to couple to asterilant source (not shown for the sake of clarity). In somevariations, the sterilant port may comprise an inlet and outlet. In somevariations, the sterilant port may optionally comprise one or more of acheck valve and particle filter configured to reduce contamination intothe sterilant port when not connected to a robot or actuator. The firstconnector 1710 may comprise a first alignment feature 1760 such as a setof protrusions on the first distal end 1714 of the first connector 1710.The alignment features may ensure that small positioning errors due torobotic manipulation do not impact the operation of the fluid connector.

The fluid connector 1700 may further comprise a second connector 1720including a second proximal end 1722 and a second distal end 1724. Thesecond proximal end 1722 may be configured to couple to the second fluiddevice (not shown for the sake of clarity). The second proximal end 1722may comprise a Luer connector or any other suitable connector. Thesecond distal end 1724 may include a second port 1726 and second housing1727. The second housing 1727 is shown in FIG. 17A holding the secondport 1726 in the closed configuration. The second connector 1720 maycomprise a second alignment feature 1762 such as a set of holes on thesecond distal end 1724 of the second connector 1720. The secondalignment feature 1762 may be configured to couple to the firstalignment feature 1760 in a predetermined axial and rotationalconfiguration to aid mating of the first connector 1710 and the secondconnector 1720.

The first port 1716 and the second port 1726 retained within respectivefirst housing 1717 of the first distal end 1714 and second housing 1727of the second distal end 1724 facilitates robotic control as the ports1716, 1726 are not separable from the fluid connector 1700, andtherefore reduces the risk of failure of automated handling by a robot.

In some variations, the first connector 1710 may comprise a first robotengagement feature 1770 and the second connector 1720 may comprise asecond robot engagement feature 1772. The robot engagement features1770, 1772 may be configured to be manipulated by a robot (e.g., robot1540) such a robotic arm. In some variations, the robot engagementfeatures 1770, 1772 may be operatively coupled to a respective firstport 1716 and second port 1726 and configured to actuate the ports 1716,1726 between a closed port configuration and an open port configuration,as shown in FIGS. 17A-17F. Additionally or alternatively, a user maymanually actuate the robot engagement features 1770, 1772 to actuaterespective ports 1716, 1726.

FIG. 17D is a front perspective view of the fluid connector 1700 in anopen port configuration. FIG. 17E is a rear perspective view and FIG.17F is a rear view of the fluid connector 1700 in the open portconfiguration. In the open port configuration, the first valve 1718 ofthe first connector 1710 and the second valve 1728 of the secondconnector 1720 are shown in FIG. 17D.

FIG. 18A is a side view and FIG. 18B is a cross-sectional side view of afluid connector 1800 in an uncoupled configuration. In some variations,the fluid connector 1800 may comprise a first connector 1810 including afirst housing 1817 comprising a first port 1816, a sterilant port 1850configured to couple to a sterilant source (not shown), a firstalignment feature 1860 configured to couple to a corresponding alignmentfeature (not shown) of the second connector 1820. The fluid connector1800 may comprise a second connector 1820 including a second housing1827 comprising a second port 1826. The first connector 1810 and secondconnector 1820 may be axially aligned and alignment features may aidrotational alignment of the first connector 1810 to the second connector1820. The first valve 1818 may comprise a first valve stem 1819 and thesecond valve 1828 may comprise a second valve stem 1829.

FIG. 18C is a side view and FIG. 18D is a cross-sectional side view ofthe fluid connector 1800 in a coupled configuration where the firsthousing 1817 and the second housing 527 are brought together but wherethe first connector 1810 and the second connector 1820 are not in fluidcommunication since the first port 1816 and the second port 1826 areboth in the closed configuration. The first alignment features on eachconnector 1810, 1820 may be configured to ensure axial and/or rotationalalignment between the first connector 1810 and the second connector1820.

FIG. 18E is a side view and FIG. 18F is a cross-sectional side view ofthe fluid connector 1800 in an open port configuration. Each of thefirst port 1817 and the second port 1827 are transitioned from theclosed configuration to an open configuration. This creates a closedinternal volume within respective distal ends of each connector 1810,1820. Each of first valve 1818 and second valve 1828 is in a closedconfiguration such that fluid flow is inhibited between the firstconnector 1810 and the second connector 1820. still restricted on eachhalf on account of the auto-shutoff valves in both sides.

FIG. 18G is a side view and FIG. 18H is a cross-sectional side view ofthe fluid connector 1800 in an open valve configuration where the firstvalve 1818 is coupled to the second valve 1828. For example, the secondvalve 1828 may be translated along a longitudinal axis of the secondconnector 1820 towards the first valve 1818. As shown in FIGS. 18G and18H, the second connector 1820 may be axially compressed to translatethe second valve 1828 towards the first valve 1818. The first valve 1818coupled to the second valve 1828 may form a radial seal, and the firstvalve stem 1819 and the second valve stem 1829 may be in contact toenable fluid communication between the first connector 1810 and thesecond connector 1820.

FIGS. 19-26B are schematic diagrams of variations of fluid connectorsystems for coupling fluid devices. In some variations, a fluidconnector may comprise a first connector configured to couple to any oneof a plurality of second connectors. FIG. 19 is a schematic diagram ofan illustrative variation of a fluid connector system 1900 comprising afirst connector 1910, a plurality of second connectors 1920, 1921, 1922,a first fluid device 1930 (e.g., sterile liquid transfer device), asecond fluid device 1940 (e.g., consumable), and a robot 1960 (e.g.,robotic arm, 3DOF robot). The first connector 1910 may be coupled influid communication with the first fluid device 1930, and the secondconnectors 1920, 1921, 1922 may be coupled in fluid communication withthe second fluid device 1940. The first connector 1910 and the secondconnectors 1920, 1921, 1922 may each comprise a port 1916 configured tocouple to a corresponding port as described in more detail herein. Therobot 1960 may comprise one or more end effectors 1962, 1964 configuredto manipulate and/or couple to one or more of the first fluid device1930 and first connector 1910. For example, the first connector 1910 maycomprise one or more sterilization ports 1950 configured to couple to anend effector 1962 (e.g., gripper). Similarly, the first fluid device1930 may comprise one or more fluid ports 1952 configured to couple toan end effector 1964.

In some variations, the robot 1960 may be configured to couple to one ormore of a sterilant source, fluid source, and pump in order tofacilitate efficient and shared fluidic connections between the fluiddevice, fluid connector, and a sterilization system. For example, FIG.96A is a plan view of a fluid device 9600 (e.g., sterile liquid transferdevice) comprising a fluid port 9610 configured to couple to a fluidsource (not shown) and a sterilization port 9620 configured to couple toa sterilant source (not shown). The FIGS. 96B and 96C are respectiveside and perspective views of a fluid device 9600 coupled to a robot9650. In some variations, the robot 9650 may comprise one or more fluidconduits 9660 configured to couple to one or more of the fluid port 9610and sterilization port 9620 of the fluid device 9600.

In some variations, a fluid connector may comprise a third connectordisposed between a first connector and a second connector. FIG. 20A is aschematic diagram of an illustrative variation of a fluid connectorsystem 2000 comprising a first connector 2010, a plurality of secondconnectors 2020, 2021, 2022, a third connector 2070 (e.g., instrument,sterilization enclosure), a first fluid device 2030 (e.g., sterileliquid transfer device), a second fluid device 2040 (e.g., consumable),and a robot 2060 (e.g., robotic arm, 3 DOF robot, 1 DOF robot). Thefirst connector 2010 may be coupled in fluid communication with thefirst fluid device 2030, and the second connectors 2020, 2021, 2022 maybe coupled in fluid communication with the second fluid device 2040. Thethird connector 2070 may be coupled between the first connector 2010 andone of the second connectors 2020, 2021, 2022. The third connector 2070may comprise a lumen configured to receive and circulate a sterilantthrough one or more portions of the first connector 2010, secondconnector 2020, 2021, 2022, and third connector 2070. In somevariations, the sterilization port 2052 may be non-removably coupled toa sterilant source and/or fluid source, thereby simplifying one or moreof the first fluid device 2030 and first connector 2010.

The robot 2060 may comprise one or more end effectors 2062, 2064, 2066configured to manipulate and/or couple to one or more of the first fluiddevice 2030, first connector 2010, and third connector 2070. Forexample, the first fluid device 2030 may comprise one or more fluidports 2050 configured to couple to an end effector 2062. Similarly, thethird connector 2070 may comprise one or more sterilization ports 2052configured to couple to robot 2060 (e.g., end effector 2064). In somevariations, the robot 2060 may be configured to couple to one or more ofa sterilant source, fluid source, and pump in order to facilitateefficient and shared fluidic connections between the fluid device, fluidconnector, and a sterilization system.

FIGS. 20B and 20C are schematic diagrams of a fluid connector connectionprocess. In FIG. 20B, a third connector 2070 may be coupled to a distalend of a first connector 2010, at 2002. A distal end of the secondconnector 2020 may be coupled to the third connector 2070, at 2004. Thesecond connector 2020 may be translated through the third connector 2070to directly couple the second connector 2020 to the first connector2010, at 2006.

In FIG. 20C, a third connector 2070 may be coupled to a distal end ofthe first connector 2010 and a distal end of the second connector 2020,at 2002. Each of the first connector 2010 and the second connector 2020may be translated toward each other through the third connector 2070, at2005. The second connector 2020 may be further translated towards thefirst connector 2010 to directly couple the first connector 2010 to thesecond connector 2010, at 2007. FIG. 20C further illustrates a firstport 2090 and a second port 2092 that may transition between a closedport configuration and an open port configuration.

In some variations, a fluid connector may comprise a third connectordisposed between a first connector and a second connector. The thirdconnector may be coupled to a second robot different from a first robotcoupled to the first connector. FIG. 21 is a block diagram of anillustrative variation of a fluid connector system 2100 comprising afirst connector 2110, a plurality of second connectors 2120, 2121, 2122,a third connector 2170 (e.g., instrument, sterilization enclosure), afirst fluid device 2130 (e.g., sterile liquid transfer device), a secondfluid device 2140 (e.g., consumable), a first robot 2160, and a secondrobot 2166. The first connector 2110 may be coupled in fluidcommunication with the first fluid device 2130, and the secondconnectors 2120, 2121, 2122 may be coupled in fluid communication withthe second fluid device 2140. The third connector 2170 may be coupledbetween the first connector 2110 and one of the second connectors 2120,2121, 2122. The third connector 2170 may comprise a lumen configured toreceive and circulate a sterilant through one or more portions of thefirst connector 2110, second connector 2120, 2121, 2122, and thirdconnector 2170. In some variations, the third connector 2170 may benon-removably coupled to a sterilant source and/or fluid source, therebysimplifying one or more of the first fluid device 2130 and firstconnector 2110.

The first robot 2160 may comprise one or more end effectors 2162, 2164configured to manipulate and/or couple to one or more of the first fluiddevice 2130 and first connector 2110. For example, the first fluiddevice 2130 may comprise one or more fluid ports 2150 configured tocouple to an end effector 2162. The third connector 2170 may be coupledto a second robot 2166 (e.g., 3 DOF robot). In some variations, therobot 2160, 2166 may be configured to couple to one or more of asterilant source, fluid source, and pump in order to facilitateefficient and shared fluidic connections between the fluid device, fluidconnector, and a sterilization system.

In some variations, a fluid connector may comprise a sterilant sourcecoupled to a plurality of second connectors. FIG. 22 is a block diagramof an illustrative variation of a fluid connector system 2200 comprisinga first connector 2210, a plurality of second connectors 2220, 2221,2222, a first fluid device 2230 (e.g., sterile liquid transfer device),a second fluid device 2240 (e.g., consumable), a robot 2260, a sterilantsource 2290 comprising one or more valves, and a sterilant switch 2292.The first connector 2210 may be coupled in fluid communication with thefirst fluid device 2230, and the second connectors 2220, 2221, 2222 maybe coupled in fluid communication with the second fluid device 2240. Therobot 2260 may comprise one or more end effectors 2262, 2264 configuredto manipulate and/or couple to one or more of the first fluid device2230 and first connector 2210. For example, the first fluid device 2230may comprise one or more fluid ports 2250 configured to couple to an endeffector 2262. In some variations, the sterilant source 2290 may becoupled to the switch 2292. The switch 2292 may be coupled to each ofthe second connectors 2220, 2221, 2222 in order to facilitate efficientand shared fluidic connections between the fluid device, fluidconnector, and sterilization system. In some variations, a sterilantconduit may be routed from the switch 2292 through the second fluiddevice 2240 to a respective second connector 2220, 2221, 2222.

In some variations, a fluid device may comprise one or more sterilantvalves coupled to a plurality of second connectors. FIG. 23 is a blockdiagram of an illustrative variation of a fluid connector system. FIG.23 is a block diagram of an illustrative variation of a fluid connectorsystem 2300 comprising a first connector 2310, a plurality of secondconnectors 2320, 2321, 2322, a first fluid device 2330 (e.g., sterileliquid transfer device), a second fluid device 2340 (e.g., consumable),a robot 2360, a set of sterilant valves 2390 disposed within a housingof the second fluid device 2340, and a sterilant switch 2392. The firstconnector 2310 may be coupled in fluid communication with the firstfluid device 2330, and the second connectors 2320, 2321, 2322 may becoupled in fluid communication with the second fluid device 2340. Therobot 2360 may comprise one or more end effectors 2362, 2364 configuredto manipulate and/or couple to one or more of the first fluid device2330 and first connector 2310. For example, the first fluid device 2330may comprise one or more fluid ports 2350 configured to couple to an endeffector 2362. In some variations, the sterilant valves 2390 may becoupled to the switch 2392. The switch 2392 may be coupled to each ofthe second connectors 2320, 2321, 2322 via the sterilant valves 2390 inorder to facilitate efficient and shared fluidic connections between thefluid device, fluid connector, and sterilization system. In somevariations, a sterilant conduit may be routed from the switch 2392through the second fluid device 2340 to a respective second connector2320, 2321, 2322.

In some variations, a fluid connector may comprise a sterilant sourcecoupled to a plurality of second connectors each having a sterilant port(e.g., sterilant valve) and a sterilant conduit through a fluid device.FIG. 24A is a block diagram of an illustrative variation of a fluidconnector system 2400 comprising a first connector 2410, a plurality ofsecond connectors 2420, 2421, 2422, a first fluid device 2430 (e.g.,sterile liquid transfer device), a second fluid device 2440 (e.g.,consumable), a robot 2460, and a sterilant switch 2492 coupled to asterilant source (not shown). The first connector 2410 may be coupled influid communication with the first fluid device 2430, and the secondconnectors 2420, 2421, 2422 may be coupled in fluid communication withthe second fluid device 2440. The robot 2460 may comprise one or moreend effectors 2462, 2464 configured to manipulate and/or couple to oneor more of the first fluid device 2430 and first connector 2410. Forexample, the first fluid device 2430 may comprise one or more fluidports 2450 configured to couple to an end effector 2462.

In some variations, each of the second connectors 2420, 2421, 2422, maycomprise a respective sterilant port 2494, 2496, 2498 comprising a valvecoupled to a distal end of the second connector 2420, 2421, 2422. Insome variations, a sterilant conduit may be routed from the switch 2492through the second fluid device 2440 to a respective sterilant port2494, 2496, 2498. In some variations, a sterilant source (not shown) maybe coupled to the switch 2492. The switch 2492 may be coupled to each ofthe second connectors 2420, 2421, 2422 via the sterilant ports 2494,2496, 2498 in order to facilitate efficient and shared fluidicconnections between the fluid device, fluid connector, and sterilizationsystem.

FIG. 24B are schematic diagrams of a fluid connector connection process2402, 2404, 2406 where a first connector 2410 is coupled to a secondconnector 2420. For example, the sterilant port 2494 is in a closedvalve configuration when the first connector 2410 and the secondconnector 2420 are separated and uncoupled 2402. FIG. 24C is a detailedschematic diagram of the sterilant valve 2494. In some variations, thevalve 2494 may transition to an open valve configuration when the firstconnector 2410 is coupled to the second connector 2420, at 2404 and2406.

In some variations, a plurality of second connectors may comprise one ormore pneumatic sterilant valves and a sterilant path through a fluiddevice. FIG. 25A is a block diagram of an illustrative variation of afluid connector system 2500 comprising a first connector 2510, aplurality of second connectors 2520, 2521, 2522, a first fluid device2530 (e.g., sterile liquid transfer device), a second fluid device 2540(e.g., consumable), a robot 2560, and a sterilant switch 2592 coupled toa sterilant source (not shown). The first connector 2510 may be coupledin fluid communication with the first fluid device 2530, and the secondconnectors 2520, 2521, 2522 may be coupled in fluid communication withthe second fluid device 2540.

In some variations, each of the second connectors 2520, 2521, 2522, maycomprise a respective pneumatic sterilant port 2594, 2596, 2598comprising a valve coupled to a distal end of the second connector 2520,2521, 2522. In some variations, a sterilant conduit may be routed fromthe switch 2592 through the second fluid device 2540 to a respectivesterilant port 2594, 2596, 2598. In some variations, a sterilant source(not shown) may be coupled to the switch 2592. The switch 2592 may becoupled to each of the second connectors 2520, 2521, 2522 via thesterilant ports 2594, 2596, 2598 in order to facilitate efficient andshared fluidic connections between the fluid device, fluid connector,and sterilization system.

The robot 2560 may comprise one or more end effectors 2562, 2564configured to manipulate and/or couple to one or more of the first fluiddevice 2530, first connector 2510, and sterilant ports 2594, 2596, 2598.For example, the first fluid device 2530 may comprise one or more fluidports 2550 configured to couple to an end effector 2562. Similarly,sterilant ports 2594, 2596, 2598 may be configured to couple to the endeffector 2562 to pneumatically actuate the sterilant ports 2594, 2596,2598. A pneumatically actuated sterilant port may enable the sterilantconduit to be formed with a fewer number of check valves between thesterilant ports 2594, 2596, 2598 and switch 2592.

FIG. 25B are schematic diagrams of a fluid connector connection process2502 and 2504, where a first connector 2510 is coupled to a secondconnector 2520. For example, the sterilant port 2594 is in a closedvalve configuration when the first connector 2510 and the secondconnector 2520 are separated and uncoupled 2502. FIG. 25C is a detailedschematic diagram of the sterilant valve 2594. In some variations, thevalve 2594 may transition to an open valve configuration when the firstconnector 2510 is coupled to the second connector 2520 and the valve2594 is pneumatically actuated, at 2504.

Liquid Transfer Bus

Generally, to permit transfer of one or more of a cell product (that is,solution(s) containing cell product), fluids, and reagents between themodules, the modules of the cartridge may be fluidically coupled to oneanother either directly or via one or more liquid transfer buses. Insome variations, a liquid transfer bus may comprise a portion of thecartridge configured to control the flow and distribution of the cellproduct between modules and reservoirs. A liquid transfer bus maycomprise one or more of a fluid manifold, fluid conduit (e.g., tubing),and one or more valves (including but not limited to 2/2 valves, 3/2valves, 3/3 valves, 4/2 valves, and rotary selector valves).

Transfer of the cell product, reagents, or fluids within the cartridgemay be achieved by any pump or other structure that generates a pressuredifferential between fluid in one portion of the cartridge and fluid inanother portion of the cartridge. For example, the cartridge maycomprise one or more pump; the cartridge may be pre-loaded withpressurized fluid contained behind a valve; the cartridge may beconnected to a fluid source or a fluid sink. The cartridge may containone or more mechanical pumps (e.g., linear pump, peristaltic pump, gearpump, screw pump, plunger pump) or portions of a pump (i.e. the pump mayinterface with a pump actuator). External pressure may be applied to thecartridge, to tubing within the cartridge, or to a bag within thecartridge (that is, applying pressure either to the liquid in the bag orto headspace gas of the bag). In some variations, an arrangement of thecomponents of the cartridge may facilitate gravity-based fluid transferwithin the cartridge (e.g., gravity-fed pumping). Although one advantageof the disclosed variations may be reduced operator intervention, thesystems and methods of the disclosure may use manual operation in thedesigned workflow or as an adjunct to automated operation in case ofimperfect automated system operation. For example, a process step mayinclude manual intervention, such as fluid input or output. An operatormay intervene in an automated process to correct device operation, (e.g.manually compressing a bag to flush remaining fluid into the system).Fluid may comprise liquid and/or gas, as compressed gases suppliedexternally or provided in pressurized chambers may be used to generateliquid flow, e.g., transfer of solution containing a cell product fromone module to another.

In some variations, the liquid transfer bus may be configured to deliverthe cell product(s) to each of a series of modules in an order set bythe design of the cartridge, or in an order determined by operation ofthe system by the processor or processors. Similarly stated, somevariations of the cartridge may have the advantage that the order ofcell processing steps as well as the process parameters for any of thecell therapy processing steps may not be set by the cartridge but ratherare controlled by the controller. In some variations, the liquidtransfer bus may be controlled to deliver the cell product to themodules in any of various sequences, or to bypass one or more modules(e.g., by configuring the state of the valve(s) attached to the fluidicbus). In some variations, a module may be used more than once in amethod of cell processing. Optionally, the method may compriseperforming one or more wash steps. For example, a counterflowcentrifugal elutriation (CCE) module may be used more than once. In anillustrative method, the method comprises culturing the cell product ina first bioreactor module, transferring the cell product to the CCEmodule to enrich for a desired cell type, transferring the cell productto a second bioreactor module for a second culturing step, washing theCCE module using a wash solution, and transferring the cell product tothe CCE module for a second enrichment step.

In some variations, the liquid transfer bus or the liquid transfer busesmay be fluidically coupled to multiple bags or reservoirs used toprovide solutions or reagents, store cell products, or to collect wastesolutions or reagents.

In some variations, the cartridge may comprise one or more pumps, whichmay be fluidically coupled to the liquid transfer bus and/or one or moremodules. The pump(s) may include a motor operatively coupled to controlcircuits and a power source (e.g. a battery or electrical connectors foran off-cartridge power source). In some variations, the pump may bedivided into a pump on the cartridge and pump actuators on one or moreinstruments of the system. The pump may be an opening in the cartridgewith tubing arranged around the circumference of the opening andconfigured to receive a pump actuator (e.g., a peristaltic rotor). Bydividing components of the pump that contact the cell product (i.e.tubing) from components of the pump that perform operations of the cellproduct, (i.e. the pump actuator, e.g., peristaltic rotor), thecartridge may be compact and simplified. For example, FIG. 26A and FIG.26B illustrate a pump head 2610 and a pump 2610 of a cartridge in anuncoupled configuration (FIG. 13A) and a coupled configuration.

In some variations, one or more pumps 146 (e.g., fluid pump) maygenerate a predetermined fluid flow rate to circulate a sterilant and/orfluid. In some variations, a pump may comprise one or more of a positivedisplacement pump (e.g., peristaltic pump, diaphragm pump, syringepump), centrifugal pump, combinations thereof, and the like. One or morefluid sources may be coupled to the pump.

In some variations, the pump may be configured to receive a pump signal(generated by a controller) configured to circulate a sterilant for adwell time sufficient to sterilize at least a portion of a fluidconnector. For example, the pump may be configured to circulate thesterilant for at least 10 seconds. In some variations, the pump may beconfigured to receive a pump signal configured to circulate anon-sterilant gas (e.g., inert gas, air) to remove the sterilant.

In some variations, a discontinuous flow pump (e.g., peristaltic pump)may generate pulsatile flow as, for example, a tube contracts andrelaxes between rollers. In some variations, closed loop feedback from aflow sensor may be used to compensate for pulsatile flow to generate asubstantially continuous flow rate. For example, a flow sensor may becoupled to a fluid conduit to measure the flow rate. A controller mayreceive the measured flow rate and generate a pump signal to the pumpbased on a proportional correction function configured to reduce the“ripples” measured by the flow sensor. Additionally or alternatively, acontroller may apply periodic error correction to a pump signal toreduce periodic error that may be unique to each pump. For example, aflow sensor may measure and determine a periodic error of a pump. A pumpsignal comprising the periodic error correction may correspond to awaveform comprising an inverse shape of the error. The resulting pumpflow may correct for fluctuations in flow rate.

Controller

In some variations, a system 100 may comprise a controller 120 (e.g.,computing device) comprising one or more of a processor 122, memory 124,communication device, 126, input device 128, and display 130. Thecontroller 120 may be configured to control (e.g., operate) the workcell110. The controller 120 may comprise a plurality of devices. Forexample, the workcell 110 may enclose one or more components of thecontroller 120 (e.g., processor 122, memory 124, communication device126) while one or more components of the controller 120 may be providedremotely to the workcell 110 (e.g., input device 128, display 130).

Processor

The processor (e.g., processor 122) described here may process dataand/or other signals to control one or more components of the system(e.g., workcell 110, controller 120). The processor may be configured toreceive, process, compile, compute, store, access, read, write, and/ortransmit data and/or other signals. Additionally, or alternatively, theprocessor may be configured to control one or more components of adevice and/or one or more components of controller (e.g., console,touchscreen, personal computer, laptop, tablet, server).

In some variations, the processor may be configured to access or receivedata and/or other signals from one or more of workcell 110, server,controller 120, and a storage medium (e.g., memory, flash drive, memorycard, database). In some variations, the processor may be any suitableprocessing device configured to run and/or execute a set of instructionsor code and may include one or more data processors, image processors,graphics processing units (GPU), physics processing units, digitalsignal processors (DSP), analog signal processors, mixed-signalprocessors, machine learning processors, deep learning processors,finite state machines (FSM), compression processors (e.g., datacompression to reduce data rate and/or memory requirements), encryptionprocessors (e.g., for secure wireless data transfer), and/or centralprocessing units (CPU). The processor may be, for example, a generalpurpose processor, Field Programmable Gate Array (FPGA), an ApplicationSpecific Integrated Circuit (ASIC), a processor board, and/or the like.The processor may be configured to run and/or execute applicationprocesses and/or other modules, processes and/or functions associatedwith the system. The underlying device technologies may be provided in avariety of component types (e.g., metal-oxide semiconductor field-effecttransistor (MOSFET) technologies like complementary metal-oxidesemiconductor (CMOS), bipolar technologies like emitter-coupled logic(ECL), polymer technologies (e.g., silicon-conjugated polymer andmetal-conjugated polymer-metal structures), mixed analog and digital,and the like.

The systems, devices, and/or methods described herein may be performedby software (executed on hardware), hardware, or a combination thereof.Hardware modules may include, for example, a general-purpose processor(or microprocessor or microcontroller), a field programmable gate array(FPGA), and/or an application specific integrated circuit (ASIC).Software modules (executed on hardware) may be expressed in a variety ofsoftware languages (e.g., computer code), including structured text,typescript, C, C++, C#, Java®, Python, Ruby, Visual Basic®, and/or otherobject-oriented, procedural, or other programming language anddevelopment tools. Examples of computer code include, but are notlimited to, micro-code or micro-instructions, machine instructions, suchas produced by a compiler, code used to produce a web service, and filescontaining higher-level instructions that are executed by a computerusing an interpreter. Additional examples of computer code include, butare not limited to, control signals, encrypted code, and compressed code

Memory

The cell processing systems and devices described here may include amemory (e.g., memory 124) configured to store data and/or information.In some variations, the memory may include one or more of a randomaccess memory (RAM), static RAM (SRAM), dynamic RAM (DRAM), a memorybuffer, an erasable programmable read-only memory (EPROM), anelectrically erasable read-only memory (EEPROM), a read-only memory(ROM), flash memory, volatile memory, non-volatile memory, combinationsthereof, and the like. In some variations, the memory may storeinstructions to cause the processor to execute modules, processes,and/or functions associated with the device, such as image processing,image display, sensor data, data and/or signal transmission, data and/orsignal reception, and/or communication. Some variations described hereinmay relate to a computer storage product with a non-transitorycomputer-readable medium (also may be referred to as a non-transitoryprocessor-readable medium) having instructions or computer code thereonfor performing various computer-implemented operations. Thecomputer-readable medium (or processor-readable medium) isnon-transitory in the sense that it does not include transitorypropagating signals per se (e.g., a propagating electromagnetic wavecarrying information on a transmission medium such as space or a cable).The computer code (also may be referred to as code or algorithm) may bethose designed and constructed for the specific purpose or purposes. Insome variations, the memory may be configured to store any received dataand/or data generated by the controller and/or workcell. In somevariations, the memory may be configured to store data temporarily orpermanently

Input Device

In some variations, the display may include and/or be operativelycoupled to an input device 128 (e.g., touch screen) configured toreceive input data from a user. For example, user input to an inputdevice 128 (e.g., keyboard, buttons, touch screen) may be received andprocessed by a processor (e.g., processor 122) and memory (e.g., memory124) of the system 100. The input device may include at least one switchconfigured to generate a user input. For example, an input device mayinclude a touch surface for a user to provide input (e.g., fingercontact to the touch surface) corresponding to a user input. An inputdevice including a touch surface may be configured to detect contact andmovement on the touch surface using any of a plurality of touchsensitivity technologies including capacitive, resistive, infrared,optical imaging, dispersive signal, acoustic pulse recognition, andsurface acoustic wave technologies. In variations of an input deviceincluding at least one switch, a switch may have, for example, at leastone of a button (e.g., hard key, soft key), touch surface, keyboard,analog stick (e.g., joystick), directional pad, mouse, trackball, jogdial, step switch, rocker switch, pointer device (e.g., stylus), motionsensor, image sensor, and microphone. A motion sensor may receive usermovement data from an optical sensor and classify a user gesture as auser input. A microphone may receive audio data and recognize a uservoice as a user input.

In some variations, the cell processing system may optionally includeone more output devices in addition to the display, such as, forexample, an audio device and haptic device. An audio device may audiblyoutput any system data, alarms, and/or notifications. For example, theaudio device may output an audible alarm when a malfunction is detected.In some variations, an audio device may include at least one of aspeaker, piezoelectric audio device, magnetostrictive speaker, and/ordigital speaker. In some variations, a user may communicate with otherusers using the audio device and a communication channel. For example, auser may form an audio communication channel (e.g., VoIP call).

Additionally or alternatively, the system may include a haptic deviceconfigured to provide additional sensory output (e.g., force feedback)to the user. For example, a haptic device may generate a tactileresponse (e.g., vibration) to confirm user input to an input device(e.g., touch surface). As another example, haptic feedback may notifythat user input is overridden by the processor.

Communication Device

In some variations, the controller may include a communication device(e.g., communication device 126) configured to communicate with anothercontroller and one or more databases. The communication device may beconfigured to connect the controller to another system (e.g., Internet,remote server, database, workcell) by wired or wireless connection. Insome variations, the system may be in communication with other devicesvia one or more wired and/or wireless networks. In some variations, thecommunication device may include a radiofrequency receiver, transmitter,and/or optical (e.g., infrared) receiver and transmitter configured tocommunicate with one or more devices and/or networks. The communicationdevice may communicate by wires and/or wirelessly.

The communication device may include RF circuitry configured to receiveand send RF signals. The RF circuitry may convert electrical signalsto/from electromagnetic signals and communicate with communicationsnetworks and other communications devices via the electromagneticsignals. The RF circuitry may include well-known circuitry forperforming these functions, including but not limited to an antennasystem, an RF transceiver, one or more amplifiers, a tuner, one or moreoscillators, a digital signal processor, a CODEC chipset, a subscriberidentity module (SIM) card, memory, and so forth.

Wireless communication through any of the devices may use any ofplurality of communication standards, protocols and technologies,including but not limited to, Global System for Mobile Communications(GSM), Enhanced Data GSM Environment (EDGE), high-speed downlink packetaccess (HSDPA), high-speed uplink packet access (HSUPA), Evolution,Data-Only (EV-DO), HSPA, HSPA+, Dual-Cell HSPA (DC-HSPDA), long termevolution (LTE), near field communication (NFC), wideband code divisionmultiple access (W-CDMA), code division multiple access (CDMA), timedivision multiple access (TDMA), Bluetooth, Wireless Fidelity (WiFi)(e.g., IEEE 802.11a, IEEE 802.11b, IEEE 802.11g, IEEE 802.11n, and thelike), voice over Internet Protocol (VoIP), Wi-MAX, a protocol fore-mail (e.g., Internet message access protocol (IMAP) and/or post officeprotocol (POP)), instant messaging (e.g., extensible messaging andpresence protocol (XMPP), Session Initiation Protocol for InstantMessaging and Presence Leveraging Extensions (SIMPLE), Instant Messagingand Presence Service (IMPS)), and/or Short Message Service (SMS),EtherCAT, OPC Unified Architecture, or any other suitable communicationprotocol. In some variations, the devices herein may directlycommunicate with each other without transmitting data through a network(e.g., through NFC, Bluetooth, WiFi, RFID, and the like).

In some variations, the systems, devices, and methods described hereinmay be in communication with other wireless devices via, for example,one or more networks, each of which may be any type of network (e.g.,wired network, wireless network). The communication may or may not beencrypted. A wireless network may refer to any type of digital networkthat is not connected by cables of any kind. Examples of wirelesscommunication in a wireless network include, but are not limited tocellular, radio, satellite, and microwave communication. However, awireless network may connect to a wired network in order to interfacewith the Internet, other carrier voice and data networks, businessnetworks, and personal networks. A wired network is typically carriedover copper twisted pair, coaxial cable and/or fiber optic cables. Thereare many different types of wired networks including wide area networks(WAN), metropolitan area networks (MAN), local area networks (LAN),Internet area networks (IAN), campus area networks (CAN), global areanetworks (GAN), like the Internet, and virtual private networks (VPN).Hereinafter, network refers to any combination of wireless, wired,public and private data networks that are typically interconnectedthrough the Internet, to provide a unified networking and informationaccess system.

Cellular communication may encompass technologies such as GSM, PCS, CDMAor GPRS, W-CDMA, EDGE or CDMA2000, LTE, WiMAX, and 5G networkingstandards. Some wireless network deployments combine networks frommultiple cellular networks or use a mix of cellular, Wi-Fi, andsatellite communication.

Display

Image data may be output on a display e.g., display 130) of a cellprocessing system. In some variations, a display may include at leastone of a light emitting diode (LED), liquid crystal display (LCD),electroluminescent display (ELD), plasma display panel (PDP), thin filmtransistor (TFT), organic light emitting diodes (OLED), electronicpaper/e-ink display, laser display, and/or holographic display.

II. METHODS

Generally, the systems and devices described herein may perform one ormore cell processing steps to manufacture a cell product. FIG. 28 is aflowchart of a method of cell processing 2800. The method 2800 mayinclude enriching a selected population of cells in a solution (e.g.,fluid) 2802. For example, the solution may be conveyed to a CCE moduleof a cartridge via a liquid transfer bus. A robot may be operated tomove the cartridge to a CCE instrument so that the CCE module interfaceswith the CCE instrument. The CCE instrument may be operated to cause theCCE module to enrich the selected population of cells. Additionally oralternatively, the cell product may be introduced into and out of thecartridge via a sterile liquid transfer port (either manually orautomatically) for any of the steps described herein. In somevariations, the cartridge may be sterilized in a feedthrough port(either manually or automatically).

In some variations, a selected population of cells in the solution maybe washed 2804. For example, the solution may be conveyed to the CCEmodule of the cartridge via the liquid transfer bus. A robot may beoperated to move the cartridge to the CCE instrument so that the CCEmodule interfaces with the CCE instrument. The CCE instrument may beoperated to cause the CCE module to remove media from the solution,introduce media into the solution, and/or replace media in the solution.

In some variations, a population of cells in the solution may beselected 2806. For example, the solution may be conveyed to a selectionmodule of the cartridge via the liquid transfer bus. The robot may beoperated to move the cartridge to a selection instrument so that theselection module interfaces with the selection instrument. The selectioninstrument may be operated to cause the selection module to select theselected population of cells.

In some variations, a population of cells in the solution may be sorted2808. For example, the solution may be conveyed to a sorting module ofthe cartridge via the liquid transfer bus. The robot may be operated tomove the cartridge to a sorting instrument so that the sorting moduleinterfaces with the sorting instrument. The sorting instrument may beoperated to cause the sorting module to sort the population of cells.

In some variations, the solution may be conveyed to a bioreactor moduleof the cartridge via the liquid transfer bus to rest 2810. For example,the robot may be operated to move the cartridge to a bioreactorinstrument so that a bioreactor module interfaces with the bioreactorinstrument. The bioreactor instrument may be operated to cause thebioreactor module to maintain the cells at a set of predeterminedconditions.

In some variations, the cells may be expanded in the solution 2812. Forexample, the solution may be conveyed to the bioreactor module of thecartridge via the liquid transfer bus. The robot may be operated to movethe cartridge to the bioreactor instrument so that the bioreactor moduleinterfaces with the bioreactor instrument. The bioreactor instrument maybe operated to cause the bioreactor module to expand the cells bycellular replication.

In some variations, tissue may be digested by conveying an enzymereagent via the liquid transfer bus to a module containing a solutioncontaining a tissue such that the tissue releases a select cellpopulation into the solution 2814.

In some variations, a selected population of cells in the solution maybe activated by conveying an activating reagent via the liquid transferbus to a module containing the solution containing the cell product2816.

In some variations, the solution may be conveyed to an electroporationmodule of the cartridge via the liquid transfer bus and receive anelectroporation signal to electroporate the cells in the solution 2818.For example, the robot may be operated to move the cartridge to anelectroporation instrument so that the electroporation module interfaceswith the electroporation instrument. The electroporation instrument maybe operated to cause the electroporation module to electroporate theselected population of cells in the presence of genetic material.

In some variations, an effective amount of a vector may be conveyed viathe liquid transfer bus to a module containing the solution containingthe cell product, thereby transducing a selected population of cells inthe solution 2820.

In some variations, a formulation solution may be conveyed via theliquid transfer bus to a module containing the cell product to generatea finished cell product 2822. For example, the finished cell product maybe conveyed to one or more product collection bags. In some variations,finishing a cell product may comprise one or more steps of washingcells, concentrating cells, exchanging a buffer of the cells with aformulation buffer, and dosing cells in the formulation buffer inpredetermined quantities into one or more product collection bags and/orvessels.

In some variations, the cell product may be removed, either manually orautomatically, from the cartridge to harvest the cells 2824.

In some variations, the cell product may comprise one or more of animmune cell genetically engineered chimeric antigen receptor T cell, agenetically engineered T cell receptor (TCR) cell, a hematopoietic stemcell (HSC), and a tumor infiltrating lymphocyte (TIL). In somevariations, the immune cell may comprise a natural-killer (NK) cell.

Methods of cell processing may include a subset of cell processing stepsin any suitable order. For example, the method of cell processing mayinclude, in order, the enrichment step 2802, the selection step 2806,the activation step 2816, the transduction step 2820, the expansion step2812, and the harvesting step 2824. In some variations, the method ofcell processing may include, in order, the enrichment step 2802, theselection step 2806, the resting step 2810, the transduction step 2820,and the harvesting step 2824. In some variations, the method of cellprocessing may include, in order, the tissue-digestion step 2820, thewashing step 2804, the activation step 2816, the expansion step 2812,and the harvesting step 2824.

Generally, the methods described herein may offload the complex stepsperformed in cell processing operation to a set of instruments, therebyreducing the cost of the cartridge (which may be a consumable). In somevariations, the cartridge may contain the cell product (e.g., solutioncontaining cells) throughout a manufacturing process, with differentinstruments interfacing with the cartridge at appropriate times toperform one or more cell processing steps. For example, a cellprocessing step may comprise conveying cells and reagents to each of themodules within the cartridge. A set of instruments interfacing with acartridge facilitates process flexibility where a workcell may becustomized with a predetermined set of instruments for a predeterminedcell therapy product. For example, the order of cell processing stepsmay be customized for each cell product as described in more detailherein with respect to FIGS. 35-55.

In some variations, a cell product may be retained within the cartridgethroughout a manufacturing process (e.g., workflow). Additionally oralternatively, the cell product may be removed from the cartridge forone or more cell processing steps, either manually by an operator, orautomatically through a fluid connector (e.g., SLTP) or other accessports on the cartridge. The cell product may then be returned to thesame cartridge, transferred to another cartridge, or split among severalcartridges. In some variations, one or more cell processing steps may beperformed outside the cartridge. In some variations, processing withinthe workcell may facilitate sterile cell processing within thecartridge.

FIG. 29 is a flowchart of a method of cell processing and illustratescell processing steps performed on a cartridge (e.g., consumable) withina workcell including a CCE instrument module, a sterile liquid transfer(SLT) instrument module, and a bioreactor instrument module. Theconsumable may be configured to interface with any of the CCE instrumentmodule, SLT instrument module, and bioreactor instrument module toperform one or more cell processing steps. For example, a robot (oroperator) may be configured to move a cartridge between any of themodules of the workcell. A pump head in an instrument may engage theconsumable cartridge in order to convey fluids between the modules ofthe cartridge, into or out of various reservoirs in the cartridge,and/or through ports that permit reagents to be added or removed fromthe cartridge.

In some variations, the CCE instrument module may comprise a pump andcentrifuge configured to interface with a cartridge (e.g., consumable).The SLT instrument module may comprise one or more fluid connectors beconfigured to interface with one or more of a bag and bioreactor of acartridge. The bioreactor instrument module may comprise one or moresensors, temperature regulators, pumps, agitators, and the like, and beconfigured to interface with the cartridge. In some variations, the cellproduct may be contained within the cartridge throughout cellprocessing.

A method of cell processing depicted in FIG. 29 may include moving afluid (e.g., cells in solution) in a product bag to a CCE module (e.g.,rotor) of a cartridge (e.g., consumable) using a pump 2910. In somevariations, the fluid may be enriched using the CCE module 2912. Forexample, blood constituents may be collected in a waste bag 2913. Insome variations, the fluid may be washed using the CCE module 2914. Forexample, buffer may be collected in a waste bag 2915. In somevariations, media may be exchanged using the CCE module 2916. Forexample, one or more of buffer (e.g., formulation buffer) and media maybe collected in a waste bag 2917. In some variations, fluid may be movedto a bioreactor of the cartridge 2918.

In some variations, a fluid connector may fill a bag with a reagent2920. In some variations, a reagent (e.g., bead, vector) may be added toa bioreactor of a cartridge 2922. In some variations, a fluid connectorremoves waste from a bag 2924. In some variations, a fluid connector mayoptionally remove a sample from a bioreactor.

In some variations, cells may be moved to a bioreactor 2930. In somevariations, the cells may undergo activation or genetic modification2932. In some variations, the cells may undergo incubation 2934. In somevariations, the cells may undergo perfusion using a pump 2936. Forexample, spent media may be collected in a waste bag 2937. In somevariations, cells may undergo expansion 2938. In some variations, cellsmay be harvested after media exchange 2940.

FIG. 30A is a flowchart of a method of cell processing for autologousCART cells or engineered TCR cells. The method 3000 may comprise thesteps of enrichment, selection, activation, genetic modification,expansion, harvest/formulation, and cryopreservation. FIG. 30B is aflowchart of a method of cell processing for allogeneic CAR T cells orengineered TCR cells. The method 3010 may comprise the steps ofenrichment, activation, genetic modification (e.g., transduction,transfection), alpha/beta T cell depletion, expansion,harvest/pool/formulation, and cryopreservation.

FIG. 31 is a flowchart of a method of cell processing for hematopoieticstem cell (HSC) cells. The method 3100 may comprise the steps ofenrichment, selection, rest, genetic modification, harvest/formulation,and cryopreservation.

FIG. 32 is a flowchart of a method of cell processing for tumorinfiltrating lymphocyte (TIL) cells. The method 3200 may comprise thesteps of tissue digestion, washing, selection, activation, expansion,harvest/formulation, and cryopreservation.

FIG. 33 is a flowchart of a method of cell processing for naturalkilling (NK) CAR cells. The method 3300 may comprise the steps ofenrichment, selection, activation, genetic modification, expansion,harvest/formulation, and cryopreservation.

FIGS. 34A-34C are flowcharts of methods of cell processing forregulatory T (Treg) cells. The method 3400 may comprise the steps ofenrichment, selection, harvest/formulation, cryopreservation. The method3402 may comprise the steps of enrichment, selection, activation,genetic modification, expansion, selection (optionally),harvest/formulation, and cryopreservation. The method 3404 may comprisethe steps of introducing feeder cell culture for enrichment, selection,activation/expansion, and harvest/irradiation. Another set of cells mayundergo enrichment, selection, co-culture with the processed feedercells, harvest, and cryopreservation.

FIGS. 98-101 are flowcharts of methods of cell processing for celltherapy workflows comprising split (e.g., parallel) processing. Themethod 9800 may comprise the steps of enrichment, selection, activation,genetic modification, expansion, formulation, and cryopreservation. Forexample, a cell processing method 9800 (e.g., workflow) may comprisesplitting a cell product into two or more portions after an enrichmentstep. The split portions may be processed in parallel within a singlecartridge. In some variations, one or more split portions may betransferred to two or more cartridges and processed in parallel. One ormore cell processing parameters (e.g., timing of process steps, types ofreagents added, transfection constructs, and the like) may be configuredindependently for each split portion of the cell product. In somevariations, the split portions may be pooled after the expansion step.

The method 9900 may comprise the steps of enrichment, selection,activation, genetic modification, expansion, formulation, andcryopreservation. For example, a cell processing method 9900 (e.g.,workflow) may comprise splitting a cell product into two or moreportions after an activation step. The split portions may be processedin parallel within a single cartridge. In some variations, one or moresplit portions may be transferred to two or more cartridges andprocessed in parallel. One or more cell processing parameters (e.g.,timing of process steps, types of reagents added, transfectionconstructs, and the like) may be configured independently for each splitportion of the cell product. In some variations, the split portions maybe pooled after the expansion step and/or the genetic modification step.

The method 10000 may comprise the steps of enrichment, selection,activation, genetic modification, expansion, formulation, andcryopreservation. For example, a cell processing method 10000 (e.g.,workflow) may comprise splitting a cell product into two or moreportions aftera selection step. The split portions may be processed inparallel within a single cartridge. In some variations, one or moresplit portions may be transferred to two or more cartridges andprocessed in parallel. One or more cell processing parameters (e.g.,timing of process steps, types of reagents added, transfectionconstructs, and the like) may be configured independently for each splitportion of the cell product. In some variations, the split portions maynot be pooled.

The method 10100 may comprise the steps of enrichment, selection,activation, genetic modification, expansion, formulation, andcryopreservation. For example, a cell processing method 10100 (e.g.,workflow) may comprise splitting a cell product into two or moreportions as starting materials. The separate products may remainsegregated and processed in parallel as split portions within a singlecartridge or a plurality of cartridges. One or more cell processingparameters (e.g., timing of process steps, types of reagents added,transfection constructs, and the like) may be configured independentlyfor each split portion. In some variations, the split portions may bepooled after the expansion step.

FIG. 102 is a schematic diagram of a cell processing system 10200configured for split processing within a single cartridge. For example,the methods 9800-10100 described with respect to FIGS. 98-101 may beperformed within the cartridge 10210. In some variations, the system10200 may comprise a sterile liquid transfer device 10220 comprising areagent 10222, and a cartridge 10210 comprising a plurality ofbioreactor modules 10230, a pump module 10240, a thermal module 10245, apressure driven flow module 10250, a MACS module 10255, anelectroporation module 10260, a FACS module 10265, a CCE module 10270,and a blank module 10280. The cartridge 10210 may further comprise areagent storage 10285, a plurality of product bags 10290, and a liquidtransfer bus 10295. The liquid transfer bus 10295 may be configured tocouple the components of the cartridge 10210 for fluid communication.

In some variations, loading and removing of cell product into and out ofthe cartridge may be performed in the system or outside the system. Insome variations, the cartridge is loaded bedside to the patient or donorand then delivered to a cell processing system in or near the hospital,or shipped to a facility where the cell processing system is installed.Likewise, the cell product may be removed from the cartridge afterprocessing either at a facility or closer to the intended recipient ofthe cell product (the patient). Optionally the cell product is frozenbefore, during, or after the methods of the disclosure—optionally afteraddition of one or more cryoprotectants to the cell product. In somevariations, the system comprises a freezer and/or a liquid nitrogensource. In some variations, the system comprises a water bath or awarming chamber containing gas of controlled temperature to permitcontrolled thawing of the cell product, e.g. a water bath set to betweenabout 20° C. and about 40° C. In some variations, the cartridge is madeof materials that resistant mechanical damage when frozen.

Automated Cell Processing

Described here are methods of transforming user-defined cell processingoperations into cell processing steps using the automated cellprocessing systems and devices described herein. In some variations,cell processing operations are received and transformed into cellprocessing steps to be performed by the system given a set ofpredetermined constraints. For example, a user may input a set ofbiologic process steps and corresponding biologic process parameters tobe executed by a cell processing system. Optionally, process parametersmay be customized for each cartridge or sets of cartridges.

FIG. 35 is a flowchart that generally describes a variation of a methodof automated cell processing. The method 3500 may include receiving anordered input list of cell processing operations 3502. For example, aset of more than one ordered input list of cell processing operationsmay be received to be performed on more than one cartridge on anautomated cell processing system. For example, as shown in the GUI 4900of FIG. 49 and described in more detail herein, one or more biologicprocess inputs (e.g., available operations) such as enrichment, MACSselection, activation, transduction, transfection, expansion, and inlineanalysis may be selected as an ordered input list of cell processingoperations. Furthermore, GUI 5200 of FIG. 52 illustrates a completeordered input list of cell processing operations (e.g., set of selectedoperations) 5220 selected by a user.

In some variations, one or more sets of cell processing parameters maybe received 3504. Each set of cell processing parameters may beassociated with one of the cell processing operations. Each set of cellprocessing parameters may specify characteristics of the cell processingstep to be performed by the instrument at that cell processing step. Forexample, the GUI 4000 of FIG. 40 illustrates reagent and containerparameters, the GUI 4200 of FIG. 42 illustrates an example of a processparameter, the GUI 4400 of FIG. 44 illustrates an example of apreprocess analytic, and the GUI 4800 of FIG. 48 illustrates an exampleof a set of activation settings.

In some variations, a transformation model may be executed on theordered input list 3506. In some variations, the transformation modelmay comprise constraints on the ordered output list determined by apredetermined configuration of the automated cell processing system. Forexample, the constraints may comprise information on the configurationof the automated cell processing system.

In some variations, the constraints may comprise one or more of a typeand/or number and/or state of instruments, a type and/or number and/orstate of modules on the cartridge, a type and/or number of reservoirs onthe cartridge, a type and/or number of sterile liquid transfer ports onthe cartridge, and number and position of fluid paths between themodules, reservoirs, and sterile liquid transfer ports on the cartridge.

In some variations, a set of predetermined constraints may be placed ona set of the process control parameters. For example, the volume and/orthe type of reagents used may be constrained based on the size of thesystem and/or products manufactured. Other process parameter constraintsmay include, but is not limited to, one or more or temperature, volume,time, pH, cell size, cell number, cell density, cell viability,dissolved oxygen, glucose levels, volumes of onboard reagent storage andwaste, combinations thereof, and the like. For example, the GUI 4000 ofFIG. 40 depicts that a reagent has a volume per unit of 30 ml and arequired volume of 54 ml, and a consumable container has a volume perunit of 75 ml. The GUI 4800 of FIG. 48 depicts that an activationconcentration is 12 mg/L, an activation culture time is 1600 seconds,activation temperature is 18° C., and a gas mix includes 21% oxygen,78.06% nitrogen, and 0.04% of carbon dioxide. These constraints may beapplied by a transformation model to generate an ordered output list ofcell processing steps that affect how one or more of the robot,instrument, and cartridge are operated and the cell productmanufactured.

In some variations, the order of operations may be constrained based onhardware constraints. For example, the robot may be limited to movingone cartridge at a time. Similarly, an instrument may be constrained tooperating on a predetermined number of cartridges at once.

In some variations, as illustrated in the GUI 4900 of FIG. 49, a loadproduct operation must be the first operation performed, and may beperformed once for each process. A fill and finish operation may alwaysbe the last operation performed before product completion, and may beperformed once for each process.

In some variation, the system may prevent the user from executing a setof operations in an order that cannot be performed by the system.

In some variations, a notification (e.g., warning, alert) may be outputif a user orders a set of operations in a “non-standard” manner. Forexample, a notification may be output if the same type of operation isrepeated sequentially (e.g., enrichment immediately followed byenrichment). Similarly, a notification may be output if an operation(e.g., selection, activation) is used two times or more within a givenprocess when such an operation is typically used just once in a givenprocess.

In some variations, an output of the transformation model may correspondto an ordered output list of cell processing steps capable of beingperformed by the system 3508. For example, the transformation model maybe executed on the sets of ordered input lists to create the orderedoutput list of cell processing steps. The output list of cell processingsteps may control a robot, cartridge, and one or more instruments.

In some variations, the ordered output list is performed by the systemto control a robot to move one or more cartridges each containing a cellproduct between the instruments 3510. For example, the MACS selectionprocess selected by the user may correspond to the robot 230 of FIG. 2moving the cartridge 250 to the cell selection instrument 216 from, forexample, another instrument. In some variations, the ordered output listmay comprise instructions for a robot to load a cartridge (e.g., singleuse consumable) into the cell processing system (e.g., workcell).Furthermore, the robot may be configured to move the cartridge to afirst instrument position.

In some variations, the ordered output list is further performed by thesystem to control one or more of the instruments to perform one or morecell processing steps on one or more cell products 3512 of a respectivecartridge. For example, the compute server rack 210 (e.g., controller120) may be configured to control an electroporation module 220configured to apply a pulsed electric field to a cell suspension of acartridge 250. In some variations, the ordered output list may compriseinstructions for an instrument (e.g., bioreactor) to process the product(e.g., transfer the cell product from a small bioreactor module to alarge bioreactor module). Furthermore, the instrument may be furtherconfigured to operate under a set of process parameters (e.g., 9 hourduration, pH of 6.7, temperature between 37.3° C. and 37.8° C., mixingmode 3). As another example, the ordered output list may compriseinstructions to operate a sterile liquid transfer module to perform oneor more of removing waste from a cartridge, adding media to thecartridge, and adding a MACS reagent to the cartridge.

In some variations, one or more electronic batch records may begenerated 3514 based on the process parameters and data collected fromsensors during process execution. Batch records generated by the systemmay include process parameters, time logging, sensor measurements fromthe instruments, QC parameters determined by QC instrumentation, andother records.

FIG. 36 is a flowchart that generally describes a variation of a methodof executing a transformation model 3600. In some variations, one ormore biological functions may be generated and output to a user. Forexample, a set of configurable biological function blocks may bedisplayed on a graphical user interface for user selection. The GUI mayenable a user to select and order the biological function blocks anddefine biological control parameters. One or more control parameters ofthe biologic function blocks may be modified by a user if desired. Insome variations, one or more biologic function templates may begenerated comprising a predefined sequence of biological functionblocks. One or more biological control parameters of the biologicfunction templates may be modified by a user if desired.

In some variations, a cell processing system may be configured toreceive and/or store one or more biologic function (e.g., process)inputs from the user 3604. For example, a user may select one or morepredefined biological function templates.

In some variations, a biologic process model (e.g., process definition)may be generated based on the biologic process inputs 3606. In somevariations, a biologic process model may include one or more ofenrichment, isolation, MACS selection, FACS selection, activation,genetic modification, gene transfer, transduction, transfection,expansion, formulation (e.g., harvest, pool), cryopreservation, T celldepletion, rest, tissue digestion, washing, irradiation, co-culture,combinations thereof, and the like.

In some variations, the biologic process model may be transformed intoan instrument execution process model 3608. For example, each biologicalfunction block in the biological process model may correspond to anordered list of cell processing system operations with correspondinghardware control parameters. The instrument execution process model maycomprise the sequence of hardware operations corresponding to thebiologic process model. As described herein, the transformation modelmay comprise one or more constraints.

Optionally, in some variations, a cell processing system may beconfigured to receive and/or store one or more instrument executionprocess inputs from the user 3610. For example, a user may modify thetransformed instrument execution process model if desired. The user mayselect specific hardware components to perform certain steps, modifytiming parameters, and the like.

In some variations, the instrument execution process may be executed togenerate the cell product 3612. For example, the cell processing systemat run-time may process the cell product through the system as definedby the instrument execution process model.

In some variations, an instrument execution process may be executed3612. In some variations, an instrument execution process model may betransformed back into a biologic process model 3614. This progress ofthe biologic process model may be output (e.g., displayed) to a user formonitoring. For example, the instrument execution process model maycomprise one or more references (e.g., pointers) back to the biologicalprocess model so that run-time execution progress may be reportedagainst the biological process model.

In some variations, a cell product may be monitored 3616. For example,the GUIs 5300 and 5400 of respective FIGS. 53 and 54 illustrate sensordata monitored by the system for a plurality of products. For example, anumber of viable cells and a status of a process (e.g., as a function ofpercentage completion) may be graphically illustrated for a user.

In some variations, an electronic record may be generated based on themonitored data 3618. For example, one or more electronic batch recordsmay be generated in compliance with, for example, 21 CFR regulations.

FIG. 55 is a block diagram of an illustrative variation of amanufacturing workflow 5500 comprising a processing platform 5520 (e.g.,system 100, workcell 110, 200, 201) configured to generate a pluralityof cell products (e.g., first product, second product, third product) inparallel. For example, a first workflow 5510 for a first product mayinclude a plurality of biologic processes 5512 executed in apredetermined sequence using corresponding elements 5522 (e.g.,hardware) of the platform 5520. Simultaneously, a second workflow 5530for a second product may execute a predetermined sequence of biologicprocesses 5530 using corresponding elements 5524 of the platform 5520.In this manner, hardware resources of the platform 5520 may beefficiently utilized to increase throughput. In some variations, about2, 3, 4, 5, 6, 7, 8, 9, 10, or more cell products may be manufacturedsimultaneously on the platform 5520. The transformation model mayinclude hardware constraints that eliminate scheduling conflicts toensure that, for example, the same instrument is not used for differentproducts at the same time.

Graphical User Interface

In some variations, a graphical user interface (GUI) may be configuredfor designing a process and monitoring a product. FIG. 37 is a variationof a GUI 3700 comprising an initial process design interface. Forexample, GUI 3700 may be a process design home page. The GUI 3700 mayindicate that no processes have been selected or loaded. A create icon3710 (e.g., “Create a Process”) may be selectable for a user to begin aprocess design process. In some variations, one or more of the GUIsdescribed herein may include a search bar.

FIG. 38 is a variation of a GUI 3800 relating to creating a process. GUI3800 may be displayed following selection of the create icon 3710 inFIG. 37. For example, GUI 3800 may comprise a process creation window3810 allowing a user to input and/or select one or more of a processname, process description, and template. In some variations, a user mayselect from a list of predetermined templates. For example, a user maycreate a process and save it as a template for later selection.

FIG. 39 is a variation of a GUI 3900 comprising relating to an emptyprocess. GUI 3900 may be displayed following confirmation in GUI 3800that a process is to be created. GUI 3900 may indicate the process name(e.g., Car T Therapy) and may highlight Process Setup icon 3910 andallow process specific parameters to be added such as process reagentsand containers, process parameters, and preprocess analytics. GUI 3900may further comprise an Add Process Reagents and Containers icon 3920,Add Process Parameters icon 3930, and Add Preprocess Analytics icon3940. Once process setup is completed, one or more process elements maybe specified.

In some variations, the GUI 3900 may comprise one or more predeterminedtemplates for a set of biological processes (e.g., CAR-T, NK cells, HSC,TIL, etc.). For example, the templates may aid process development andbe validated starting points for process development. The templates maybe further modified (e.g., customized) based on user requirements.

FIG. 40 is a variation of a GUI 4000 comprising relating to adding areagent and a consumable container. GUI 4000 may be displayed followingselection of an Add Process Reagents and Containers icon 3920 in FIG.39. For example, GUI 4000 may comprise an Add Reagent and Containerwindow 4010 enabling a user to input and/or select one or more reagentscomprising a reagent kind, manufacturer, part number, volume per unit,required volume and required reagent inputs (e.g., lot number,expiration date, requires container transfer). Add Reagent and Containerwindow 4010 may comprise one or more of an input field, selection box,drop-down selector, and the like. Furthermore, the Add Reagent andContainer window 3810 may enable a user to input and/or select one ormore consumable containers comprising a manufacturer, part number,volume per unit, and required container inputs (e.g., lot number,expiration date). In some variations, a user may select from a list ofpredetermined templates. For example, a user may create a process andsave it as a template.

FIG. 41 is a variation of a GUI 4100 comprising relating to a processparameter. GUI 4100 may be displayed following selection of an AddProcess Reagents and Containers icon 3930 in FIG. 39. For example, GUI4100 may comprise an Add Process Parameter window 4110 enabling a userto input and/or select one or more parameters comprising a name,parameter identification, description, data type, units, and parametertype. Add Process Parameter window 4010 may comprise one or more of aninput field, selection box, drop-down selector, and the like. In somevariations, a user may select from a list of predetermined templates.For example, a user may create a parameter and save it as a template.FIG. 42 is a variation of a GUI 4200 comprising relating to a patientweight process parameter. For example, GUI 4200 may comprise an AddProcess Parameter window 4110 having filled in parameter informationincluding patient weight, data type (e.g., integer), units (e.g., kg),and parameter type (e.g., input).

FIG. 43 is a variation of a GUI 4300 relating to a preprocess analytic.GUI 4300 may be displayed following selection of an Add Preprocessanalytics icon 3940 in FIG. 39. For example, GUI 4300 may comprise anAdd Preprocess Analytic window 4310 enabling a user to input and/orselect one or more parameters comprising a name, identifier,description, data type, and display group. Add Preprocess Analyticwindow 4310 may comprise one or more of an input field, selection box,drop-down selector, and the like. In some variations, a user may selectfrom a list of predetermined templates. For example, a user may create aparameter and save it as a template.

FIG. 44 is a variation of a GUI 4400 relating to a white blood cellcount preprocess analytic. For example, GUI 4400 may comprise an AddPreprocess Analytic window 4410 having filled in preprocess analyticinformation including name (e.g., CBC White Blood Cell Count),identifier (e.g., CBC-white-blood-cell-count), description (e.g., Numberof white blood cells in a sample), data type (e.g., float), and displaygroup (e.g., WBC).

FIG. 45 is a variation of a GUI 4500 relating to a process parametercalculation. GUI 4500 may be displayed following selection of an AddPreprocess analytics icon 3940 in FIG. 39 and selection of a“Calculation” parameter type. For example, GUI 4500 may comprise an AddPreprocess Analytic window 4510 enabling a user to input and/or selectone or more parameters comprising a name, identifier, description, datatype, display group, units, and parameter type. Furthermore, aCalculation Builder may enable a user to define a formula (e.g.,algorithm, equation) to perform a predetermined calculation. Forexample, a Calculation Builder may comprise one or more of a set ofavailable parameters (e.g., patient weight), constant value, equation,and operands.

FIG. 46 is a variation of a GUI 4600 relating to a completed processsetup. For example, GUI 4600 may comprise a Process Setup window 4610having a filled in process reagents, containers, process parameters, andpreprocess analytics. Once process setup is completed, one or moreprocess elements may be specified.

FIG. 47 is a variation of a GUI 4700 relating to process operationsactivation settings. GUI 4700 may be displayed following selection of aProcess elements icon 4620 in FIG. 46. For example, GUI 4700 maycomprise an Activation settings window 4710 allowing a user to inputand/or select one or more of activation concentration (e.g., mg/L),activation culture time (e.g., seconds), activation temperature (e.g., °C.), and gas mix mode. In some variations, a user may select from a listof predetermined templates. For example, a user may create a set ofactivation settings and save it as a template for later selection.

FIG. 48 is a variation of a GUI 4800 relating to a filled processoperations activation settings. For example, GUI 4800 may comprise anActivation settings window 4810 having filled in Activation settinginformation. In some variations, a set of gases (e.g., O₂, N₂, CO₂) andcorresponding concentrations may be specified.

FIG. 49 is a variation of a GUI 4900 relating to a process operationsinterface. The GUI 4900 may comprise an Available Operations window 4910and a Selected Operations window 4920. The available options forselection may include one or more biologic process inputs as describedherein including, but not limited to, enrichment, MACS selection,activation, transduction, transfection, expansion, and inline analysis.One or more of the operations may be selected and dragged into theSelected Operations window 4920. The selected operations may bereordered within the Selected Operations window 4920.

FIG. 50 is a variation of a GUI 5000 relating to dragging processoperations. The GUI 5000 may comprise an Available Operations window5010, a Selected Operations window 5020, and a selected (e.g., dragged)operation 5030 that may be drag and dropped between the AvailableOperations window 5010 and the Selected Operations window 5020. TheSelected Operations window 5020 may comprise a plurality of selectedoperations.

FIG. 51 is a variation of a GUI 5100 relating to dragging processoperations. The GUI 5100 may comprise an Available Operations window5110, a Selected Operations window 5120, and a selected (e.g., dragged)operation 5130 that may be drag and dropped between the AvailableOperations window 5110 and the Selected Operations window 5120. TheSelected Operations window 5120 may comprise a plurality of selectedoperations.

FIG. 52 is a variation of a GUI 5200 relating to a filled processoperations. For example, the GUI 5200 may comprise an AvailableOperations window 5210 and a Selected Operations window 5220 comprisinga completed set of selected operations. In some variations, the settings(e.g., parameters) of each operation may be selectively modified by theuser by selecting a corresponding icon (e.g., gear icon).

FIGS. 53 and 54 are variations of a GUI 5300 and 5400 relating toproduct monitoring. The GUI 5300 and 5400 may comprise respectivemonitoring windows 5310, 5410. For example, the GUI 5310 may monitor aplurality of products 5320 and output one or more productcharacteristics 5330 including, but not limited to, a summary, processdata, online analytics, imaging, process audit logs, process parameters,and process schedule. The monitoring window 5410 may monitor one or moreproduct characteristics of one or more products. For example, theproduct characteristics may include, but is not limited to, one or moreof a process name, identification, process identification, progress,estimated completion, current step, and message.

FIG. 77A is a flowchart of a method of separating cells 7700 using a CCEmodule. FIG. 77B is a flowchart of a method of concentrating cells 7710using a CCE module. FIG. 77C is a flowchart of a method of bufferexchange 7720 using a CCE module.

FIG. 78 is a flowchart of a method of separating cells 7800. A method ofcounterflow centrifugal elutriation (CCE) 7800 may comprise the stepmoving a rotor towards a magnet 7802. The rotor may define a rotationalaxis. In some variations, moving the rotor comprises advancing andwithdrawing the magnet relative to the rotor using a robot. The rotormay be optionally moved towards an illumination source and an opticalsensor 7804. Fluid may be flowed through the rotor 7806. In somevariations, flowing the fluid comprises a flow rate of up to about 150ml/min while rotating the rotor. The rotor may be magnetically rotatedabout the rotational axis using the magnet while flowing the fluidthrough the rotor 7808. In some variations, rotating the rotor comprisesa rotation rate of up to 6,000 RPM. One or more of the fluid and thecells may be optionally illuminated using an illumination source 7810.Image data of one or more of the fluid and biological material (e.g.,particles, cellular material) in the rotor may optionally be generatedusing an optical sensor 7812. One or more of a rotation rate of therotor and a flow rate of the fluid may optionally be selected based atleast in part on the image data 7814. The fluid may be flowed out of therotor 7816. The rotor may be moved away from the magnet 7818. The rotormay optionally be moved away from the illumination source and theoptical sensor 7820.

FIG. 79A is a flowchart of a closed-loop method of separating cells7900. FIG. 79B is a flowchart of a closed-loop method of elutriatingcells 7910. FIG. 79C is a flowchart of a closed-loop method ofharvesting cells 7920.

FIG. 80A is a flowchart of a method of separating cells 8000. FIG. 80Bis a flowchart of a method of selecting cells 8010.

FIG. 81 is a flowchart of a method of separating cells 8100. A method ofmagnetic-activated cell selection (MACS) may comprise labeling cellswith a reagent 8102. In some variations, a magnetic-activated cellselection (MACS) reagent may be incubated with the input cells to labelthe set of cells with the MACS reagent. In some variations, incubatingthe MACS reagent comprises a temperature between about 1° C. and about10° C. The fluid comprising input cells may be flowed into a flow cell8104. A set of the cells are labeled with the MACS reagent. In somevariations, the magnet array may optionally be moved relative to theflow cell 8106. In some variations, the set of cells may be magneticallyattracted towards a magnet array for a dwell time 8108. In somevariations, the dwell time may be at least about one minute. In somevariations, the magnet array may be disposed external to the flow cell.In some variations, a longitudinal axis of the flow cell isperpendicular to ground. In some variations, the flow cell may be absentbeads. In some variations, the magnet array may optionally be moved awayfrom the flow cell to facilitate flowing the set of cells out of theflow cell 8110. The set of cells may be flowed out of the flow cellafter the dwell time 8112. For example, flowing the set of cells out ofthe flow cell may comprise flowing a gas through the flow cell. Thefluid without the set of cells may optionally be flowed out of the flowcell after the dwell time 8114.

FIG. 82A is a flowchart of a method of preparing a bioreactor 8200. FIG.82B is a flowchart of a method of loading a bioreactor 8210. FIG. 82C isa flowchart of a method of preparing a bioreactor 8220. FIG. 82D is aflowchart of a method of calibration for a bioreactor 8230. FIG. 82E isa flowchart of a method of mixing reagents 8240. FIG. 82F is a flowchartof a method of mixing reagents 8250. FIG. 82G is a flowchart of a methodof culturing cells 8260. FIG. 82H is a flowchart of a method ofrefrigerating cells 8270. FIG. 82I is a flowchart of a method of takinga sample 8270. FIG. 82J is a flowchart of a method of culturing cells8280. FIG. 82K is a flowchart of a method of media exchange 8290. FIG.82L is a flowchart of a method of controlling gas 8292. FIG. 82M is aflowchart of a method of controlling pH 8294.

FIG. 83 is a flowchart of a method of electroporating cells 8300 usingan electroporation module. In some variations, an electroporation modulemay comprise a fluid conduit configured to receive a first fluidcomprising cells and a second fluid, a set of electrodes coupled to thefluid conduit, a pump coupled to the fluid conduit, and a controllercomprising a processor and memory.

A method of electroporating cells may optionally comprise generating afirst signal to introduce the first fluid into the fluid conduit usingthe pump 8302. A first fluid comprising cells in a fluid conduit may bereceived 8304. In some variations, a second signal may optionally begenerated to introduce the second fluid into the fluid conduit such thatthe second fluid separates the first fluid from a third fluid 8306. Insome variations, the second fluid may comprise a gas or oil. A secondfluid in the fluid conduit may be received to separate the first fluidfrom a third fluid 8308. An electroporation signal may optionally begenerated to electroporate the cells in the fluid conduit using the setof electrodes 8310. An electroporation signal may be applied to thefirst fluid to electroporate the cells 8312. In some variations, thefirst fluid may be substantially static when applying theelectroporation signal. In some variations, a third signal mayoptionally be generated to introduce the third fluid into the fluidconduit 8314. The third fluid may be separated from the first fluid bythe second fluid. The third fluid may optionally be received in thefluid conduit separated from the first fluid by the second fluid 8316.

FIG. 84 is a flowchart of a method of electroporating cells 8400. Amethod of electroporating cells may comprise receiving a first fluidcomprising cells in a fluid conduit 8402. A resistance measurementsignal may be applied to the first fluid using a set of electrodes 8404.A resistance may be measured between the first fluid and the set ofelectrodes 8406. An electroporation signal may be applied to the firstfluid based on the measured resistance 8408. In some variations, asecond fluid comprising a gas may optionally be received in the fluidconduit before applying the electroporation signal to the fluid. Thefirst fluid may be separated from a third fluid by the second fluid.

Fluid Connector

A method of transferring fluid using a fluid connector 2700 is describedin the flowchart of FIG. 27 and illustrated schematically in thecorresponding steps depicted in FIGS. 16B-16L. The method 2700 maycomprise the step of coupling a sterilant source to a fluid connector2702. For example, as shown in FIG. 16B, the inlet 1652 and outlet 1654is coupled to a sterilant source to form a fluid pathway or connection.In some variations, a robot may be configured to couple and decouple thesterilant source to the sterilant port 1650 using a fluid conduit suchas a tube. In some variations, the fluid connector 1600 may comprise aplurality of sterilant ports 1650. As described herein, in somevariations, a sterilant port may optionally comprise one or more of acheck valve and a particle filter configured to reduce ingress of debris(e.g., after disconnecting the fluid connector). In some variations, thesterilant source may comprise or be coupled to a pump configured tocirculate a sterilant through the sterilant port 1650. In somevariations, the sterilant port 1650 may be coupled to one or more of asterilant source and a fluid source such as a heated air source. Forexample, a first sterilant port may be configured to couple to a firststerilant source, a second sterilant port may be configured to couple toa second sterilant source, and a third sterilant source may beconfigured to couple to an air source.

The separate portions of the fluid connector 1600 may be broughttogether and mated. The method 2700 may comprise coupling a first portof a first connector to a second port of a second connector 2704. FIG.16C is a schematic diagram of the fluid connector 1600 where the firstport 216 and second port 226 are in a coupled configuration (e.g. dockedposition) that forms a first seal. In some variations, the firstconnector 1610 and the second connector 1620 may be axially and/orrotationally aligned, and one or more of the connectors 1610, 1620 maybe translated to couple the connectors 1610, 1620 together. In FIG. 16C,the first port 1616 and the second port 1626 are each in a closedconfiguration where the lumens of the respective first connector 1610and second connector 1620 are sealed from the external environment tomaintain sterility of the lumen of the fluid connector 1600.Furthermore, the first valve 1618 and the second valve 1628 are each ina closed configuration that seals the proximal and distal ends of theconnectors from each other. For example, the first valve 1618 in theclosed configuration forms a seal (e.g., barrier) between the firstproximal end 1612 and the first distal end 1614. Similarly, the secondvalve 1628 in the closed configuration forms a seal between the secondproximal end 1622 and the second distal end 1624. In this manner, evenif a portion of a connector is contaminated (e.g., first distal end1614), then the other portions of the fluid connector 1600 (e.g., firstproximal end 1612, second connector 1620) may remain sterile by virtueof one or more of the port seals and valve seals.

The ports may be transitioned to an open configuration such that adistal end of the connectors may be in fluid communication. The method2700 may comprise transitioning the ports to an open configuration 2706.FIG. 16D is a schematic diagram of the fluid connector 1600 where thefirst port 1616 and the second port 1626 are transitioned into an openport configuration to create a shared volume between the valves 1618,1628 that is isolated from the external environment. In FIG. 16D, thefirst valve 1618 and the second valve 1628 are in the closedconfiguration such that the chamber 1615 defines the volume (e.g.,cavity) of the fluid connector 1600 between the first valve 1618 and thesecond valve 1628. That is, the first distal end 1614 is in fluidcommunication with the second distal end 1624. The ports 1616, 1627 maybe received and/or held in respective housings 1617, 1627 in the closedconfiguration. In some variations, a robot may be configured totransition the ports 1616, 1626 between the open configuration and theclosed configuration as described in more detail herein. Additionally oralternatively, the first port 1616 and second port 1626 mayautomatically transition (e.g., mechanically actuate) from the closedconfiguration to the open configuration upon mating the first port 1616to the second port 1626.

In some variations, a fluid may be flowed into the fluid connector toaid sterilization. The method 2700 may comprise flowing fluid (e.g.,liquid, gas) into the fluid connector through the sterilant port 2708.FIG. 16E is a schematic diagram of the fluid connector 1600 where thefirst chamber 1615 receives a fluid such as air at a predeterminedtemperature, pressure, and/or humidity. In some variations, one or moreportions of the fluid connector 1600 may be dehumidified. For example,pressurized hot air may optionally be circulated within chamber 1615 inorder to remove residual fluid, moisture, and raise a temperature of theinner surfaces of the chamber 1615. The circulated fluid may flowthrough housings 1617, 1627 and over inner and/or outer surfaces of theports 1616, 1626.

Generally, sterilization of a fluid connector may comprise one or moresteps of dehumidification, conditioning, decontamination, and aeration(e.g., ventilation). Dehumidification may include removing moisture fromthe fluid connector. Conditioning may include heating the surfaces ofthe fluid connector to be decontaminated in order to preventcondensation and aid sterilization. Decontamination may includecirculating a sterilant through the fluid connector at a predeterminedconcentration, rate, and exposure time. Aeration may include removingthe sterilant from the fluid connector by circulating a gas (e.g.,sterile air) through the fluid connector.

A sterilant may be flowed into the fluid connector to sterilize one ormore portions of the fluid connector. As described in more detailherein, the sterilant may be, for example, vaporized hydrogen peroxide(VHP) and/or ionized hydrogen peroxide (IHP). The method 2700 maycomprise flowing a sterilant into the fluid connector through thesterilant port 2710. FIG. 16F is a schematic diagram of the fluidconnector 1600 where the first chamber 1615 receives the sterilant for apredetermined amount of time (e.g., dwell time). For example, thesterilant may be circulated within the chamber 1615 to sterilize thechamber 1615 of the fluid connector 1600 and any contents disposedtherein (e.g., other fluid, biological material). In some variations,the dwell time may be up to about 10 minutes, and between about 1 minuteto about 10 minutes, including all ranges and sub-values in-between. Insome variations, the vaporized hydrogen peroxide may comprise aconcentration between about 50% and about 70%, including all ranges andsub-values in-between. Additionally or alternatively, one or more of thefirst valve 1618 and the second valve 1628 may be in the openconfiguration such that the sterilant may be circulated through otherportions of the fluid connector 1600 such as first proximal end 1612 andsecond proximal end 1622.

In some variations, the valves may be translated relative to each other.The method 2700 may comprise translating a first valve relative to asecond valve 2712. FIG. 16G is a schematic diagram of the fluidconnector 1600 where the first valve 1618 and second valve 1628 arecoupled to each other (e.g., transfer position). The first valve 1618coupled to the second valve 1628 forms a second seal between the firstconnector 1610 and the second connector 1620.

The valves may be transitioned to an open configuration such that eachend of the fluid connector is in fluid communication. The method 2700may comprise transitioning the first valve and the second valve from aclosed configuration to an open configuration 2714. In some variations,the first valve and the second valve may comprise a spring-loadedshutoff configured to actuate to the open configuration, therebyallowing for fluidic communication between the sterile lumens of thefirst connector 1610 and the second connector 1620. In some variations,each of the first valve 1618 of a first connector 1610 and the secondvalve 1628 of a second connector 1620 may comprise an engagement featuresuch as threading configured to facilitate coupling between the firstvalve 1618 and the second valve 1628. For example, once the second valve1628 is translated to contact the first valve 1618, the engagementfeatures of the valves 1618, 1628 may be coupled (e.g., locked) byrotating (e.g., twisting) one of the first valve 1618 and the secondvalve 1628 to engage their respective threads to each other. Conversely,one of the first valve 1618 and the second valve 1628 may be rotated inthe opposite direction to uncouple (e.g., unlock) the first valve 1618from the second valve 1628.

In some variations, fluid may flow through the fluid connector 2716.FIG. 16H is a schematic diagram of the fluid connector depicted in FIG.16A transferring fluid between fluid devices coupled to the fluidconnector. For example, the contents (e.g., fluid, biological material)of the first fluid device 1630 and the second fluid device 1640 may betransferred through the fluid connector 1600. In some variations, one ormore of a pump, gravity feed, and the like may aid transfer through thefluid connector 1600.

In some variations, another fluid may be flowed into the fluid connectorafter fluid transfer between a first fluid device and a second fluiddevice has been completed. The method 2700 may comprise flowing fluid(e.g., liquid, gas, sterilant) into the fluid connector through thesterilant port 2708 to remove a fluid and/or biological material fromthe fluid connector 2718. For example, flowing an inert gas into thefluid connector may reduce drops of liquid from forming when the firstconnector and second connector are separated. If a sterilant is flowedthrough the fluid connector, another fluid such as an inert gas may beflowed to aerate the fluid connector and ensure that the sterilant isremoved.

To begin decoupling the fluid connector, the valves may be translatedaway from each other. The method 2700 may comprise decoupling the firstconnector and the second connector 2720. In some variations, a robot maybe configured to manipulate the fluid connector 1600 to transition thevalves 1618, 1628 to a closed configuration and to translate the valves1618, 1628 away from each other, which may occur simultaneously orindependently. The valves 1618, 1628 in the closed configuration inhibitfluid flow between the first connector 1610 and the second connector1620. FIG. 16I is a schematic diagram of the fluid connector 1600 in aclosed valve configuration where the second valve 1628 is translatedaway from the first valve 1618. Accordingly, the fluid connector 1600returns to the docked position. For example, the first valve 1618 andthe second valve 1628 may be configured to engage their respectivespring-loaded shutoff features to form a seal and reduce drips and/orleaks. In some variations, one or more of a fluid and sterilant mayoptionally be configured to circulate through the chamber 1615 to removemoisture and/or sterilize the chamber 1615.

FIG. 16J is a schematic diagram of the fluid connector 1600 where thefirst port 1616 and the second port 1626 are transitioned from the openport configuration to the closed port configuration. In some variations,a robot may be configured to manipulate the fluid connector 1600 totransition the ports 1616, 1626 to a closed position to seal a lumen ofthe first connector 1610 from a lumen of the second connector 1620. Insome variations, the ports 1616, 1626 may be configured to automaticallytransition to the closed port configuration when the first valve 1618separates from the second valve 1628.

FIG. 16K is a schematic diagram of the fluid connector 1600 where thesecond connector 1620 is translated away from the first connector 1610.In some variations, a robot may be configured to manipulate the fluidconnector 1600 to separate the first connector 1610 from the secondconnector 1620. FIG. 16K depicts the fluid connector 1600 in adisengaged configuration.

FIG. 16L is a schematic diagram of the fluid connector 1600 decoupledfrom the sterilant source. In some variations, a robot may be configuredto manipulate the fluid connector 1600 and/or sterilant source toseparate the sterilant source 1650 from the sterilant source. In somevariations, the sterilant source may be decoupled from the fluidconnector 1600 at any point after completing a sterilization process.

In some variations, the cartridge comprises one or more Sterile LiquidTransfer Ports (SLTPs) configured for use with a Sterile Liquid TransferDevice (SLTD). In some variations, the SLTP comprises one or more of acap, a fitting, and a tube fluidically coupled to the fitting. The capmay be removable or pierceable. The fitting may be a push-to-connectfitting (PTCF) or a threaded fitting. PTCF include male-to-female,female-to-male, and androgynous fittings. Illustrative SLTPs and SLTDssuitable for use in the systems of the disclosure may include, forexample, AseptiQuik® S connectors, Lynx® CDR connectors, Kleenpak™connectors, Intact™ connectors, GE LifeScience® ReadyMate connectors.

When the disclosure refers to sterile liquid transfer devices, sterileliquid transfer ports, and sterile liquid transfer, the word “sterile”should be understood as a non-limiting description of some variations—anoptional feature providing advantages in operation of certain systemsand methods of the disclosure. Maintaining sterility is typicallydesirable for cell processing but may be achieved in various ways,including but not limited to providing sterile reagents, media, cells,and other solutions; sterilizing cartridge(s) and/or cartridgecomponent(s) after loading (preserving the cell product fromdestruction); and/or operating the system in a sterile enclosure,environment, building, room, or the like. Such operator performed orsystem performed sterilization steps may make the cartridge or cartridgecomponents sterile and/or preserve the sterility of the cartridge orcartridge components.

III. EXAMPLES

FIGS. 85-96D are diagrams of other variations of a fluid connector. FIG.85 depicts a fluid connector 8500 comprising a first connector 8510including a first cap 8516 and a second connector 8520 including asecond cap 8526. Fluid connector 8500 may comprise a male connector anda female connector, each with a removable cap and internal self-shutoffvalve configured to reduce leaks and drips. The first cap 8516 and thesecond cap 8526 may be removable from their respective connectors 8510,8520.

In some variations, the fluid connector may be used with aself-sterilizing cap and decap tool 8600 depicted in FIG. 86. Thecap/decap tool 8600 may be configured to facilitate a sterileenvironment (e.g., ISO5) where the caps may be removed and theconnectors pressed together, first sealing the connectors to each other,and then pressed further to transition the internal self-shutoff valvesto an open configuration.

In some variations, the tool 8600 may be configured to remove andre-apply caps to the fluid connector 8500, and to provide a sterilevolume for aseptic connection and disconnection of the fluid connector8500 pair. In some variations, a method of using the tool 8600 maycomprise inserting both capped connectors in a first configuration(e.g., where the caps approach the closed shutters) such that the fluidconnectors form a seal within a lumen of the decap tool 8600. In somevariations the shutters may be opened to ensure a decap mechanism isretracted. Both capped connectors may be pushed to form a secondconfiguration. The decap mechanism may be engaged to lock into featureson the caps. Both capped connectors may be retracted to the firstconfiguration where the caps are retained in the decap mechanism. Thedecap mechanism may be retracted such that the caps are held within arecess in the tool 8600. The internal volume may optionally bedecontaminated with sterilant or heat. Both connectors may be advancedto connect and perform the transfer. The steps described herein may besequentially reversed.

FIG. 87 depict a coupling sequence for a self-sealing fluid connector8700 comprising a first connector 8710 and a second connector 8720. Thefluid connector 8700 may be configured to reduce leaks and drips and mayfacilitate smoother fluid flow path by removing spring elements fromcontact with fluid.

FIG. 88 depict a coupling sequence for a self-sealing fluid connector8800 comprising a first connector 8810 and a second connector 8820.

In some variations, a fluid connector may transfer fluids in a sterilemanner using a retractable needle. FIG. 89 depicts a fluid connector8900 comprising a first connector 8910 and a second connector 8920. Thefirst connector 8910 may comprise a first cap 8916 configured toremovably couple to a distal end of the first connector 8910. The firstconnector 8910 may comprise a first elastomeric member 8970 (e.g.,sealing septum) and a first thermal member 8972 (e.g., thermallyresealable septum) disposed at a distal end of the first connector 8910.The first connector 8910 may further comprise a needle 8990 and a spring8992 coupled to the first elastomeric member 8970 and the needle 8990.The second connector 8920 may comprise a second cap 8926 configured toremovably couple to a distal end of the second connector 8920. Thesecond connector 8920 may comprise a second elastomeric member 8980(e.g., sealing septum) and a second thermal member 8982 (e.g., thermallyresealable septum) disposed at a distal end of the second connector8920.

In some variations, the needle 8990 may be advanced through each of thefirst elastomeric member 8970, first thermal member 8972, second thermalmember 8982, and second elastomeric member 8980 to form a fluid pathwaybetween the first connector 8910 and the second connector 8920. Fluidmay flow through the first connector 8910 and into the second connector8920 via a lumen of needle 8990. Each of the elastomeric members 8970,8980 and thermal members 8972, 8982 may seal once the needle 8990 iswithdrawn from a distal end of the first connector 8910. For example,the thermal member 8972, 8982 may be configured to thermally seal at apredetermined temperature and the elastomeric members 8970, 8980 mayself-seal once the needle 8990 has been withdrawn. In some variations,the fluid connector 8900 may be thermally decontaminated and resealedafter fluid transfer. For example, the fluid connector 8900 (e.g.,thermal members 8972, 8982) may be heated using one or more of a laser,contact heating, heated air, combinations thereof, and the like.

In some variations, a fluid connector may comprise a port comprising anactuator configured to transition the port between a closed portconfiguration and an open port configuration. In some variations, theactuator may comprise a spring such as an external spring, a rotaryspring, and a linear spring, as described in more detail with respect toFIGS. 90A-96D.

FIGS. 90A-90C depict a fluid connector having an external springactuator. FIG. 90A is a side view, FIG. 90B is a perspective view, andFIG. 90C is a cross-sectional side view of a fluid connector 9000comprising a first connector 9010 and second connector 9020. The firstconnector 9010 may comprise a first port 9016 comprising a first spring9036, and the second connector 9020 may comprise a second port 9026comprising a second spring 9046. The springs 9036, 9046 may beconfigured to actuate respective ports 9016, 9026 between a closed portconfiguration and an open port configuration. Although not shown in FIG.90C, springs 9036, 9046 may be coupled in an extended configuration tothe pin in the open port configuration.

FIGS. 91A-91F depict a fluid connector having a linear spring actuator.FIG. 91A is a side view, FIG. 91B is a perspective view, and FIG. 91C isa cross-sectional side view of the fluid connector 9100 in an open portconfiguration. The fluid connector 9100 may comprise a first connector9110 and second connector 9120. The first connector 9110 may comprise afirst port 9116 comprising a first spring 9136, and the second connector9120 may comprise a second port 9126 comprising a second spring 9146.The springs 9136, 9146 may be configured to actuate respective ports9116, 9126 between a closed port configuration and an open portconfiguration. FIG. 91D is a side view, FIG. 91E is a perspective view,and FIG. 91F is a cross-sectional side view of the fluid connector 9100in a closed configuration.

FIGS. 92A-92D depict a fluid connector having a rotary spring actuator.FIG. 92A is a side view, FIG. 92B is a transparent side view, FIG. 92Cis a perspective view, and FIG. 92D is a cross-sectional side view of afluid connector 9200 comprising a first connector 9210 and secondconnector 9220. The first connector 9210 may comprise a first port 9216comprising a first spring 9236, and the second connector 9220 maycomprise a second port 9226 comprising a second spring 9246. The springs9236, 9246 may be configured to actuate respective ports 9216, 9226between a closed port configuration and an open port configuration. FIG.92B shows the ports 9216, 9226 in an open port configuration and FIG.92D shows the ports 9216, 9226 in a closed port configuration.

FIGS. 93A-94B depict fluid connectors having ports enclosed within ahousing (e.g., enclosure). FIG. 93A is a perspective view and FIG. 93Bis a transparent perspective view of a fluid connector 9300 comprising afirst connector 9310 having a first housing 9338 and first actuator9336, and a second connector 9320 having a second housing 9348 and asecond actuator 9346. FIG. 93B shows a first port 9316 enclosed withinfirst 9338 housing. The first port 9316 is coupled to the first actuator9336 configured to transition the first port 9316 between an open portconfiguration (shown in FIG. 93B) and a closed port configuration.

FIG. 94A is a perspective and FIG. 94B is a transparent perspective viewof a fluid connector 9400 comprising a first connector 9410 having afirst housing 9438 and a first actuator 9436, and a second connector9420 having a second housing 9448 and a second actuator 9446. FIG. 94Bshows a first port 9416 enclosed within first 9438 housing. The firstactuator 9436 coupled to the first port 9416 may be configured totransition the first port 9416 between an open port configuration (shownin FIG. 94B) and a closed port configuration.

FIG. 95A is a perspective view and FIG. 95B is a transparent perspectiveview of a fluid connector 9500 comprising a first connector 9510 havinga first housing 9538, first port 9516, and a first actuator 9536. Asecond connector 9520 may comprise a second housing 9548, second port9526, and a second actuator 9546. FIG. 95B shows the first port 9516 andthe second port 9526 each in an open port configuration. For example,the first actuator 9536 coupled to the first port 9516 may be configuredto transition the first port 9516 between an open port configuration anda closed port configuration. FIG. 95C is a detailed side view of thefirst port 9516 and first actuator 9536 in an open port configuration,and FIG. 95D is a detailed side view of the first port 9516 and firstactuator 9536 in a closed port configuration.

FIG. 97A is a perspective view of a MACS module. FIG. 97B is across-sectional perspective view of a MACS module. FIG. 97C is across-sectional side view of a MACS module.

As used herein, sterile should be understood as a non-limitingdescription of some variations, an optional feature providing advantagesin operation of certain systems and methods of the disclosure.Maintaining sterility is typically desirable for cell processing but maybe achieved in various ways, including but not limited to providingsterile reagents, media, cells, and other solutions; sterilizingcartridge(s) and/or cartridge component(s) after loading (preserving thecell product from destruction); and/or operating the system in a sterileenclosure, environment, building, room, or the like. Such user or systemperformed sterilization steps may make the cartridge or cartridgecomponents sterile and/or preserve the sterility of the cartridge orcartridge components.

All references cited are herein incorporated by reference in theirentirety.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

All embodiments of any aspect of the disclosure can be used incombination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” “below,” and words of similarimport, when used in this application, shall refer to this applicationas a whole and not to any particular portions of the application.

While embodiments of the present invention have been shown and describedherein, those skilled in the art will understand that such embodimentsare provided by way of example only. Numerous variations, changes, andsubstitutions will now occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed in practicing the invention. It is intended that the followingclaims define the scope of the invention and that methods and structureswithin the scope of these claims and their equivalents be coveredthereby.

1.-237. (canceled)
 238. A fluid connector, comprising: a first connectorcomprising a first proximal end configured to couple to a first fluiddevice, and a first distal end comprising a first port; and a secondconnector comprising a second proximal end configured to couple to asecond fluid device, and a second distal end comprising a second portconfigured to couple to the first port, wherein the first distal endcomprises a first lumen and a first valve, the first valve configured totranslate within the first lumen; and the second distal end comprises asecond lumen and a second valve, the second valve is configured totranslate within the second lumen, and one of the first valve and thesecond valve is configured to translate within the first lumen and thesecond lumen.
 239. The fluid connector of claim 238, wherein the firstvalve and the second valve are configured to transition from a closedconfiguration to an open configuration only when the first valve couplesto the second valve.
 240. The fluid connector of claim 238, wherein thefirst port and the second port are configured to transition between anopen configuration and a closed configuration.
 241. The fluid connectorof claim 238, wherein the first connector comprises a first portactuator and/or the second connector comprises a second port actuator.242. The fluid connector of claim 238, wherein the second port coupledto the first port defines a chamber.
 243. The fluid connector of claim242, wherein one or more of the first connector and the second connectorcomprises a sterilant port configured to couple to a sterilant source,the sterilant port configured to be in fluid communication with thefirst distal end and the second distal end when the second port iscoupled to the first port.
 244. The fluid connector of claim 243,wherein the chamber is configured to receive one or more of a fluid anda sterilant from the sterilant port.
 245. The fluid connector of claim238, wherein the sterilant port is configured to receive a sterilantsuch that the sterilant sterilizes the first connector and the secondconnector.
 246. The fluid connector of claim 238, wherein the firstconnector comprises a first valve, and the second connector comprises asecond valve configured to couple to the first valve.
 247. The fluidconnector of claim 238, wherein a first seal comprises the first portcoupled to the second port, and a second seal comprises the first valvecoupled to the second valve.
 248. The fluid connector of claim 243,wherein the sterilant comprises one or more of vaporized hydrogenperoxide, ionized hydrogen peroxide, and ethylene oxide.
 249. The fluidconnector of claim 238, further comprising one or more robot engagementfeatures.
 250. The fluid connector of claim 238, wherein the firstconnector comprises a first alignment feature and the second connectorcomprises a second alignment feature configured to couple to the firstalignment feature in a predetermined axial and rotational configuration.251. The fluid connector of claim 238, wherein one or more of the firstfluid device and the second fluid device comprises an instrument.
 252. Asystem of claim 238, further comprising: a robot configured to operatethe fluid connector; a controller comprising a memory and processor, thecontroller coupled to the robot, the controller configured to: generatea first port signal to couple the first port to the second port usingthe robotic arm.
 253. The system of claim 252, wherein the controller isconfigured to generate a first valve signal to translate the first valverelative to the second valve using the robotic arm, and generate asecond valve signal to transition the first valve and the second valveto the open configuration.
 254. The system of claim 252, wherein thecontroller is configured to generate a second port signal to decouplethe first port from the second port, wherein the sterility of the fluidconnector is maintained before coupling the first port to the secondport and after decoupling the first port from the second port.
 255. Thesystem of claim 252, further comprising a fluid pump coupled to thesterilant source, wherein the controller is configured to generate afirst fluid pump signal to circulate a fluid into the chamber throughthe sterilant port.
 256. The system of claim 255, wherein the controlleris configured to generate a second fluid pump signal to circulate thesterilant into the chamber through the sterilant port to sterilize atleast the chamber.
 257. The system of claim 255, wherein the controlleris configured to generate a third fluid pump signal to remove thesterilant from the chamber.
 258. The system of claim 252, wherein thecontroller is configured to generate a thermal sterilization signal tothermally sterilize the fluid connector.
 259. The system of claim 252,wherein the controller is configured to generate a radiationsterilization signal to sterilize the fluid connector using radiation.260. The system of claim 252, wherein the robot is configured to couplea fluid connector between at least two of the plurality of instrumentsand the cartridge.
 261. The system of claim 260, further comprising thefluid connector of claim 238, and further comprising: a controllercomprising a memory and processor, the controller coupled to the robot,the controller configured to: generate a port signal to couple the firstport to the second port using the robotic arm; generate a first valvesignal to translate the first valve relative to the second valve usingthe robotic arm; and generate a second valve signal to transition thefirst valve and the second valve to the open configuration. 262.-276.(canceled)
 277. The fluid connector of claim 238, wherein one or more ofthe first fluid device and the second fluid device comprises one or moreof a counterflow centrifugal elutriation (CCE) instrument, amagnetic-activated cell selection (MACS) instrument, an electroporationinstrument, and a bioreactor instrument.
 278. The fluid connector ofclaim 238, wherein one or more of the first fluid device and the secondfluid device comprises one or more of a counterflow centrifugalelutriation (CCE) module, a magnetic-activated cell selection (MACS)module, an electroporation module, and a bioreactor module.
 279. Thefluid connector of claim 238, wherein one or more of the first fluiddevice and the second fluid device comprise a liquid transfer bus. 280.The fluid connector of claim 238, wherein one or more of the first fluiddevice and the second fluid device comprise a reagent vessel.
 281. Amethod transferring fluid, comprising: coupling a first port of a firstconnector to a second port of a second connector; transitioning each ofthe first port and the second port from a closed configuration to anopen configuration; translating a first valve of the first connectorrelative to a second valve of the second connector; transitioning eachof the first valve and the second valve from a closed configuration toan open configuration such that the first connector is in fluidcommunication with the second connector.
 282. The method of claim 281,further comprising flowing a sterilant into the first connector and thesecond connector.